Histidine kinase

ABSTRACT

The invention provides Histidine kinase polypeptides and polynucleotides encoding Histidine kinase polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing Histidine kinase polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional PatentApplication No. 60/048,347, filed May 30, 1998.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the Histidine kinasefamily, as well as their variants, hereinafter referred to as “Histidinekinase,” “Histidine kinase polynucleotide(s),” and “Histidine kinasepolypeptide(s)” as the case may be

BACKGROUND OF THE INVENTION

[0003] The Streptococci make up a medically important genera of microbesknown to cause several types of disease in humans, including, forexample, otitis media, conjunctivitis, pneumonia, bacteremia,meningitis, sinusitis, pleural empyema and endocarditis, and mostparticularly meningitis, such as for example infection of cerebrospinalfluid. Since its isolation more than 100 years ago, Streptococcuspneumoniae has been one of the more intensively studied microbes. Forexample, much of our early understanding that DNA is, in fact, thegenetic material was predicated on the work of Griffith and of Avery,Macleod and McCarty using this microbe. Despite the vast amount ofresearch with S. pneumoniae, many questions concerning the virulence ofthis microbe remain. It is particularly preferred to employStreptococcal genes and gene products as targets for the development ofantibiotics.

[0004] The frequency of Streptococcus pneumoniae infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains which are resistantto some or all of the standard antibiotics. This phenomenon has createdan unmet medical need and demand for new anti-microbial agents,vaccines, drug screening methods, and diagnostic tests for thisorganism. Many two component signal transduction systems (TCSTS) havebeen identified in bacteria (Stock, J. B., Ninfa, A. J. & Stock, A.M.(1989) Microbiol. Rev. 53, 450-490). These are involved in thebacterium's ability to monitor its surroundings and adapt to changes inits environment. Several of these bacterial TCSTS are involved invirulence and bacterial pathogenesis within the host.

[0005] Histidine kinases are components of the TCSTS whichautophosphorylate a histidine residue. The phosphate group is thentransferred to the cognate response regulator. The Histidine kinaseshave five short conserved amino acid sequences (Stock, J. B., Ninfa, A.J. & Stock, A. M.(1989) Microbiol. Rev. 53, 450-490, Swanson, R. V.,Alex, L. A. & Simon, M. I. (1994) TIBS 19 485-491). These are thehistidine residue, which is phosphorylated, followed after approximately100 residues by a conserved asparagine residue. After another 15 to 45residues a DXGXG motif is found, followed by a FXXF motif after another10-20 residues. 10-20 residues further on another glycine motif, GXG isfound. The two glycine motifs are thought to be involved in nucleotidebinding. This family of histidine kinases includes VanS_(B) protein fromEnterococcus faecalis, V583. VanS_(B) is the histidine kinase of theTCSTS which controls the transcription of the vancomycin resistanceoperon in VanB-type enterococci. (Evers, S, & Courvalin, P., (1996), J.Bacteriol. 178, 1302-1309.)

[0006] Response regulators are components of the TCSTS. These proteinsare phosphorylated by histidine kinases and in turn once phosphorylatedeffect the response, often through a DNA binding domain becomingactivated. The response regulators are characterized by a conservedN-terminal domain of approximately 100 amino acids. The N-terminaldomains of response regulators as well as retaining five functionallyimportant residues, corresponding to the residues D12, D13, D57, T87,K109 in CheY (Matsumura, P., Rydel, J. J., Linzmeier, R. & Vacante, D.(1984) J. Bacteriol. 160, 36-41), have conserved structural features(Volz, K. (1993) Biochemistry 32, 11741-11753). The 3-dimensionalstructures of CheY from Salmonella typhimurium (Stock, A. M., Mottonen,J. M., Stock, J. B. & Schutt, ,C. E. (1989) Nature, 337, 745-749) andEscherichia coli (Volz, K. & Matsumura, P. (1991) J. Biol. Chem. 266,15511-15519) and the N-terminal domain of nitrogen regulatory protein Cfrom S.typhimurium (Volkman, B. F., Nohaile, M. J., Amy, N. K., Kustu,S. & Wemmer, D. E. (1995) Biochemistry, 34 1413-1424), are available, aswell as the secondary structure of SpoOF from Bacillus subtilis (Feher,V. A., Zapf, J. W., Hoch, J. A., Dahlquist, F. W., Whiteley, J. M. &Cavanagh, J. (1995) Protein Science, 4, 1801-1814). These structureshave a (α/β)5 fold. Several structural residues are conserved betweendifferent response regulator sequences, specifically hydrophobicresidues within the β-sheet hydrophobic core and sites from theα-helices.

[0007] Among the processes regulated by TCSTS are production ofvirulence factors, motility, antibiotic resistance and cell replication.Inhibitors of TCSTS proteins would prevent the bacterium fromestablishing and maintaining infection of the host by preventing it fromproducing the necessary factors for pathogenesis and thereby haveutility in anti-bacterial therapy.

[0008] Moreover, the drug discovery process is currently undergoing afundamental revolution as it embraces “functional genomics,” that is,high throughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on “positional cloning” and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available as well as from othersources. There is a continuing and significant need to identify andcharacterize further genes and other polynucleotides sequences and theirrelated polypeptides, as targets for drug discovery.

[0009] Clearly, there exists a need for polynucleotides andpolypeptides, such as the Histidine kinase embodiments of the invention,that have a present benefit of, among other things, being useful toscreen compounds for antibiotic activity. Such factors are also usefulto determine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists to find ways to prevent,ameliorate or correct such infection, dysfunction and disease.

[0010] Certain of the polypeptides of the invention possess significantamino acid sequence homology to a known Van S-B from Enterococcusfaecalis protein.

SUMMARY OF THE INVENTION

[0011] The present invention relates to Histidine kinase, in particularHistidine kinase polypeptides and Histidine kinase polynucleotides,recombinant materials and methods for their production. In anotheraspect, the invention relates to methods for using such polypeptides andpolynucleotides, including the treatment of microbial diseases, amongstothers. In a further aspect, the invention relates to methods foridentifying agonists and antagonists using the materials provided by theinvention, and for treating microbial infections and conditionsassociated with such infections with the identified compounds. In astill further aspect, the invention relates to diagnostic assays fordetecting diseases associated with microbial infections and conditionsassociated with such infections, such as assays for detecting Histidinekinase expression or activity.

[0012] Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following descriptions and from reading theother parts of the present disclosure.

DESCRIPTION OF THE INVENTION

[0013] The invention relates to Histidine kinase polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a Histidinekinase of Streptococcus pneumoniae, which is related by amino acidsequence homology to Van S-B from Enterococcus faecalis polypeptide. Theinvention relates especially to Histidine kinase having the nucleotideand amino acid sequences set out in Table 1 as SEQ ID NO: 1 or 3 and SEQID NO: 2 or 4 respectively. TABLE 1 Histidine kinase Polynucleotide andPolypeptide Sequences (A) Streptococcus pneumoniae Histidine kinasepolynucleotide sequence [SEQ ID NO: 1].5′-TTGGAAATTCTGGACTATCTAGTGAAMATGAAGGCCGGGCCTTGACTCGGTCTCAGATTATCGATGCCGTCTGGAAAGCGACAGATGAGGTTCCCTTTGACCGTGTTATTGATGTTTATATCAAGGAATTGCGGAAAAAGCTAGACTTGGATTGTATCCTCACTGTGCGCAATGTTGGTTATAAATTGGAGCGAAAATGAAACGAACAGGTTTATTTACAAAGATATTTATCTATACCTTCTCGATATTTAGTGTTCTGGTTATCTGCCTTCATTTAGCTATTTATTTTCTTTTTCCTTCGACTTATCTGAGTCATCGTCAGGAAACCATTGGTCAAAAGGCAACAGCCATTGCCCAGTCCCTAGAAGGGAAAGATAGGCAGAGTATCGAGCAAGTGTTAGACTTGTATTCCCAGACTAGTGATATCAAGGGGACCGTCAAAGGTGAGATGACCGAGGACAAGTTAGAAGTCAAGGACAGTCTTCCTCTGGACACAGACCGCCAGACAACCTCTCTCTTTATTGAGGAGCGCGAGGTGAAAACGCAAGACGGTGGTACTATGATTCTCCAGTTTCTAGCTTCCATGGATTTACAAAAGGAAGCGGAGCAAATCAGTCTCCAATTTCTTCCCTATACCTTGCTGGCCTCCTTTCTGATTTCCCTCTTGGTGGCCTACATCTACGCTCGGACTATTGTTGCACCGATTTTGGAAATCAAGCGGGTGACCCGTCGGATGATGGACCTGGATTCCCAAGTGCGATTGCGCGTGGATTCTAAGGATGAGATAGGCAATCTCAAGGAACAAATCAATAGCCTCTACCAGCATCTCTTGACTGTTATTGCGGACTTGCATGAAAAGAATGAAGCCATTCTCCAGCTGGAGAAGATGAAGGTCGAATTCCTACGAGGAGCTTCTCATGAATTGAAAACACCGCTGGCTAGTTTGAAAATCCTAATCGAAAATATGAGAGAGAATATCGGTCGTTATAAGGATAGAGACCAGTATCTGGGAGTTGCCTTGGGGATTGTGGATGAACTCAATCACCATGTTCTGGAGATACTTTCCCTCTCTTCTGTGCAGGAATTGCGAGATGATAGGGAAACAATTGACCTCCTCCAGATGACGCAAAATCTGGTCAAAGATTATGCCTTGCTAGCCAAGGAAAGAGAGCTCCAGATAGACAATAGTTTGACCCATCAGCAGGCTTATCTAAACCCATCAGTTATGAAGTTGATTCTTTCTAATCTCATCAGCAATGCCATTAAGCACTCTGTTCCAGGTGGCTTAGTTCGAATTGGAGAAAGAGAAGGAGAACTTTTTATCGAAAATAGCTGTAGCTCAGAGGAACAAGAAAAACTAGCCCAGTCTTTTTCTGACAATGCCAGTCGCAAGGTCAAGGGGTCTGGTATGGGGCTCTTTGTGGTTAAGAGTCTATTAGAACATGAAAAATTAGCTTATCGTTTCGAGATGGAGGAGAATAGTTTAACCTTCTTTATAGATTTTCCAAAAGTCGTCCAAGACTAG-3′ (B) Streptococcus pneumoniae Histidinekinase polypeptide sequence deduced from a polynucleotide sequence inthis table [SEQ ID NO: 2].NH₂-MKRTGLFTKTFTYTPSIPSVLVTCLHLATYPLEPSTYLSHRQETIGQKATATAQSLEGKDRQSIEQVLDLYSQTSDTKGTVKGEMTEDKLEVKDSLPLDTDRQTTSLFTEEREVKTQDGGTMILQFLASMDLQKEAEQISLQFLPYTLLASFLISLLVAYTYARIIVAPILEIKRVTRRNMDLDSQVRLRVDSKDETGNLKEQTNSLYQHLLTVTADLHEKNEATLQLEKMKVEFLRGASHELKIPLASLKTLTENMRENTGRYKDRDQYLGVALGTVDELNHHVLQILSLSSVQELRDDRETIDLLQMTQNLVKDYALLAKERELQTDNSLTHQQAYLNPSVMKLTLSNLISNATKHSVPGGLVRIGEREGELFIENSCSSEEQEKLAQSFSDNASRKVKGSGMGLFVVKSLLEHEKLAYRFEMEENSLTFFIDFPKVVQD-COOH (C) Streptococcus pneumoniae Histidinekinase ORF sequence [SEQ ID NO: 3]. 5′-CTATGAGGTGGCCCTGGTTTTACTGGATATCCAGATGCCCAAGCTTAACGGCTTAGAAGTCCTAGCTGAGATTCGTAAAACCAGTCAGGTTCCTGTCTTGATGTTGACAGCTTTTCAGGATGAGGAATACAAGATGAGTGCCTTTGCCTCTTTGGCAGATGGCTATCTGGAAAAACCTTTCTCCCTCTCCCTCTTAAAAGTGAGGGTGGACGCGATTTTCAAGCGCTACTACGATACAGGACGAATCTTTTCTTACAAGGATACCAAGGTGGACTTTGAAAGCTACAGTGCAAGCCTCGCAGGTCAAGAAGTGCCTATCAATGCCAAAGAGTTGGAAATTCTGGACTATCTAGTGAAAAATGAAGGCCGGGCCTTGACTCGGTCTCAGATTATCGATGCCGTCTGGAAAGCGACAGATGAGGTTCCCTTTGACCGTGTTATTGATGTTTATATCAAGGAATTGCGGAAAAAGCTAGACTTGGATTGTATCCTCACTGTGCGCAATGTTGGTTATAAATTGGAGCGAAAATGAAACGAACAGGTTTATTTACAAAGATATTTATCTATACCTTCTCGATATTTAGTGTTCTGGTTATCTGCCTTCATTTAGCTATTTATTTTCTTTTTCCTTCGACTTATCTGAGTCATCGTCAGGAAACCATTGGTCAAAAGGCAACAGCCATTGCCCAGTCCCTAGAAGGGAAAGATAGGCAGAGTATCGAGCAAGTGTTAGACTTGTATTCCCAGACTAGTGATATCAAGGGGACCGTCAAAGGTGAGATGACCGAGGACAAGTTAGAAGTCAAGGACAGTCTTCCTCTGGACACAGACCGCCAGACAACCTCTCTCTTTATTGAGGAGCGCGAGGTGAAAACGCAAGACGGTGGTACTATGATTCTCCAGTTTCTAGCTTCCATGGATTTACAAAAGGAAGCGGAGCAAATCAGTCTCCAATTTCTTCCCTATACCTTGCTGGCCTCCTTTCTGATTTCCCTCTTGGTGGCCTACATCTACGCTCGGACTATTGTTGCACCGATTTTGGAAATCAAGCGGGTGACCCGTCGGATGATGGACCTGGATTCCCAAGTGCGATTGCGCGTGGATTCTAAGGATGAGATAGGCAATCTCAAGGAACAAATCAATAGCCTCTACCAGCATCTCTTGACTGTTATTGCGGACTTGCATGAAAAGAATGAAGCCATTCTCCAGCTGGAGAAGATGAAGGTCGAATTCCTACAAGGAGCTTCTCATGAATTGAAA-3′ (D)Streptococcus pneumoniae Histidine kinase polypeptide sequence deducedfrom a polynucleotide ORF sequence in this table [SEQ ID NO: 4]. NH2MKRTGLFTKIFIYTFSIFSVLVICLHLAIYFLFPSTYLSHRQETIGQKATAIAQSLEGKDRQSIEQVLDLYSQTSDIKGTVKGEMTEDKLEVKDSLPLDTDRQTTSLFIEEREVKTQDGGTMILQFLASMDLQKEAEQISLQFLPYTLLASFLISLLVAYIYARTIVAPILEIKRVTRRMMDLDSQVRLRVDSKDEIGNLKEQINSLYQHLLTVIADLHEKNEAILQLEKMKVEFLQGASHELKHTAG-COOH (E) Streptococcus pneumoniaeResponse Regulator, cognate to the histidine kinase of the inventionpolynucleotide sequence [SEQ ID NO: 5].ATGAAAATTTTAATTGTAGAAGATGAAGAGATGATCCGTGAGGGGGTCAGTGATTATTTGACGGATTGTGGCTATGAAACTATTGAGGCAGCGGACGGTCAGGAAGCTCTGGAGCAATTTTCTAGCTATGAGGTGGCCCTGGTTTTACTGGATATCCAGATGCCCAAGCTTAACGGCTTAGAAGTCCTAGCTGAGATTCGTAAAACCAGTCAGGTTCCTGTCTTGATGTTGACAGCTTTTCAGGATGAGGAATACAAGATGAGTGCCTTTGCCTCTTTGGCAGATGGCTATCTGGAAAAACCTTTCTCCCTCTCCCTCTTAAAAGTGAGGGTGGACGCGATTTTCAAGCGCTACTACGATACAGGACGAATCTTTTCTTACAAGGATACCAAGGTGGACTTTGAAAGCTACAGTGCAAGCCTCGCAGGTCAAGAAGTGCCTATCAATGCCAAAGAGTTGGAAATTCTGGACTATCTAGTGAAAAATGAAGGCCGGGCCTTGACTCGGTCTCAGATTATCGATGCCGTCTGGAAAGCGACAGATGAGGTTCCCTTTGACCGTGTTATTGATGTTTATATCAAGGAATTGCGGAAAAAGCTAGACTTGGATTGTATCCTCACTGTGCGCAATGTTGGTTATAAATTGGAGCGAAAATGA (F)Steptococcus pneumoniae Response regulator polypeptide sequence, cognateof the histidine kinase of the invention deduced from a polynucleotidesequence in this table [SEQ ID NO: 6].MKILIVEDEEMIREGVSDYLTDCGYETIEAADGQEALEQFSSYEVALVLLDIQMPKLNGLEVLAETRKTSQVPVLMLTAFQDEEYKMSAEASLADGYLEKPFSLSLLKVRVDAIFKRYYDTGRIFSYKDTKVDFESYSASLAGQEVPINAKELETLDYLVKNEGRALTRSQIIDAVWKATDEVPFDRVIDVYIKELRKKLDLDCILTVRNVGYKLERK

[0014] Deposited Materials

[0015] A deposit containing a Streptococcus pneumoniae 0100993 strainhas been deposited with the National Collections of Industrial andMarine Bacteria Ltd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB21RY, Scotland on Apr. 11, 1996 and assigned deposit number 40794. Thedeposit was described as Streptococcus pneumoniae 0100993 on deposit. OnApr. 17, 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coliwas similarly deposited with the NCIMB and assigned deposit number40800. The Streptococcus pneumoniae strain deposit is referred to hereinas “the deposited strain” or as “the DNA of the deposited strain.”

[0016] The deposited strain contains the full length Histidine kinasegene. The sequence of the polynucleotides contained in the depositedstrain, as well as the amino acid sequence of any polypeptide encodedthereby, are controlling in the event of any conflict with anydescription of sequences herein.

[0017] The deposit of the deposited strain has been made under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

[0018] A license may be required to make, use or sell the depositedstrain, and compounds derived therefrom, and no such license is herebygranted.

[0019] In one aspect of the invention there is provided an isolatednucleic acid molecule encoding a mature polypeptide expressible by theStreptococcus pneumoniae 0100993 strain, which polypeptide is containedin the deposited strain. Further provided by the invention are Histidinekinase polynucleotide sequences in the deposited strain, such as DNA andRNA, and amino acid sequences encoded thereby. Also provided by theinvention are Histidine kinase polypeptide and polynucleotide sequencesisolated from the deposited strain.

[0020] Polypeptides

[0021] Histidine kinase polypeptide of the invention is substantiallyphylogenetically related to other proteins of the Histidine kinasefamily.

[0022] In one aspect of the invention there are provided polypeptides ofStreptococcus pneumoniae referred to herein as “Histidine kinase” and“Histidine kinase polypeptides” as well as biologically, diagnostically,prophylactically, clinically or therapeutically useful variants thereof,and compositions comprising the same.

[0023] Among the particularly preferred embodiments of the invention arevariants of Histidine kinase polypeptide encoded by naturally occurringalleles of the Histidine kinase gene.

[0024] The present invention further provides for an isolatedpolypeptide which:

[0025] (a) comprises or consists of an amino acid sequence which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, mostpreferably at least 97-99% or exact identity, to that of SEQ ID NO: 2over the entire length of SEQ ID NO: 2;

[0026] (b) a polypeptide encoded by an isolated polynucleotidecomprising or consisting of a polynucleotide sequence which has at least70% identity, preferably at least 80% identity, more preferably at least90% identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% or exact identity to SEQ ID NO: 1 over theentire length of SEQ ID NO: 1;

[0027] (c) a polypeptide encoded by an isolated polynucleotidecomprising or consisting of a polynucleotide sequence encoding apolypeptide which has at least 70% identity, preferably at least 80%identity, more preferably at least 90% identity, yet more preferably atleast 95% identity, even more preferably at least 97-99% or exactidentity, to the amino acid sequence of SEQ ID NO: 2, over the entirelength of SEQ ID NO: 2; or

[0028] (d) a polypeptide encoded by an isolated polynucleotidecomprising or consisting of a polynucleotide sequence which has at least70% identity, preferably at least 80% identity, more preferably at least90% identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% or exact identity, to SEQ ID NO: 1 over theentire length of SEQ ID NO: 3;

[0029] (e) a polypeptide encoded by an isolated polynucleotidecomprising or consisting of a polynucleotide sequence which has at least70% identity, preferably at least 80% identity, more preferably at least90% identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% or exact identity to SEQ ID NO: 3 over theentire length of SEQ ID NO:3; or

[0030] (f) a polypeptide encoded by an isolated polynucleotidecomprising or consisting of a polynucleotide sequence encoding apolypeptide which has at least 70% identity, preferably at least 80%identity, more preferably at least 90% identity, yet more preferably atleast 95% identity, even more preferably at least 97-99% or exactidentity, to the amino acid sequence of SEQ ID NO: 4, over the entirelength of SEQ ID NO: 4;

[0031] (g) comprises or consists of an amino acid sequence which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, mostpreferably at least 97-99% or exact identity, to the amino acid sequenceof SEQ ID NO: 2 over the entire length of SEQ ID NO: 4.

[0032] The polypeptides of the invention include a polypeptide of Table1 [SEQ ID NO: 2 or 4] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of Histidine kinase, and also those which have at least 70%identity to a polypeptide of Table 1 [SEQ ID NO: 1 or 3] or the relevantportion, preferably at least 80% identity to a polypeptide of Table 1[SEQ ID NO: 2 or 4and more preferably at least 90% identity to apolypeptide of Table 1 [SEQ ID NO: 2 or 4] and still more preferably atleast 95% identity to a polypeptide of Table 1 [SEQ ID NO: 2 or 4] andalso include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

[0033] The invention also includes a polypeptide consisting of orcomprising a polypeptide of the formula:

X −(R ₁)_(m)−(R ₂)−(R ₃)_(n) −Y

[0034] wherein, at the amino terminus, X is hydrogen, a metal or anyother moiety described herein for modified polypeptides, and at thecarboxyl terminus, Y is hydrogen, a metal or any other moiety describedherein for modified polypeptides, R₁ and R₃ are any amino acid residueor modified amino acid residue, m is an integer between 1 and 1000 orzero, n is an integer between 1 and 1000 or zero, and R₂ is an aminoacid sequence of the invention, particularly an amino acid sequenceselected from Table 1 or modified forms thereof. In the formula above,R₂ is oriented so that its amino terminal amino acid residue is at theleft, covalently bound to R₁, and its carboxy terminal amino acidresidue is at the right, covalently bound to R₃. Any stretch of aminoacid residues denoted by either R₁ or R₃, where m and/or n is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer. Other preferred embodiments of the invention are providedwhere m is an integer between 1 and 50, 100 or 500, and n is an integerbetween 1 and 50, 100, or 500.

[0035] It is most preferred that a polypeptide of the invention isderived from Streptococcus pneumoniae, however, it may preferably beobtained from other organisms of the same taxonomic genus. A polypeptideof the invention may also be obtained, for example, from organisms ofthe same taxonomic family or order.

[0036] A fragment is a variant polypeptide having an amino acid sequencethat is entirely the same as part but not all of any amino acid sequenceof any polypeptide of the invention. As with Histidine kinasepolypeptides, fragments may be “free-standing,” or comprised within alarger polypeptide of which they form a part or region, most preferablyas a single continuous region in a single larger polypeptide.

[0037] Preferred fragments include, for example, truncation polypeptideshaving a portion of an amino acid sequence of Table 1 [SEQ ID NO: 2 or4], or of variants thereof, such as a continuous series of residues thatincludes an amino- and/or carboxyl-terminal amino acid sequence.Degradation forms of the polypeptides of the invention produced by or ina host cell, particularly a Streptococcus pneumoniae, are alsopreferred. Further preferred are fragments characterized by structuralor functional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

[0038] Further preferred fragments include an isolated polypeptidecomprising an amino acid sequence having at least 15, 20, 30, 40, 50 or100 contiguous amino acids from the amino acid sequence of SEQ ID NO: 2,or an isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO: 2.

[0039] Also preferred are biologically active fragments which are thosefragments that mediate activities of Histidine kinase, including thosewith a similar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause Disease in anindividual, particularly a human.

[0040] Fragments of the polypeptides of the invention may be employedfor producing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention.

[0041] In addition to the standard single and triple letterrepresentations for amino acids, the term “X” or “Xaa” may also be usedin describing certain polypeptides of the invention. “X” and “Xaa” meanthat any of the twenty naturally occurring amino acids may appear atsuch a designated position in the polypeptide sequence.

[0042] Polynucleotides

[0043] It is an object of the invention to provide polynucleotides thatencode Histidine kinase polypeptides, particularly polynucleotides thatencode the polypeptide herein designated Histidine kinase.

[0044] In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding Histidine kinase polypeptidescomprising a sequence set out in Table 1 [SEQ ID NO: 1 or 3] whichincludes a full length gene, or a variant thereof. The Applicantsbelieve that this full length gene is essential to the growth and/orsurvival of an organism which possesses it, such as Streptococcuspneumoniae.

[0045] As a further aspect of the invention there are provided isolatednucleic acid molecules encoding and/or expressing Histidine kinasepolypeptides and polynucleotides, particularly Streptococcus pneumoniaeHistidine kinase polypeptides and polynucleotides, including, forexample, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B—and Z- DNAs. Further embodiments of the invention include biologically,diagnostically, prophylactically, clinically or therapeutically usefulpolynucleotides and polypeptides, and variants thereof, and compositionscomprising the same.

[0046] Another aspect of the invention relates to isolatedpolynucleotides, including at least one full length gene, that encodes aHistidine kinase polypeptide having a deduced amino acid sequence ofTable 1 [SEQ ID NO: 2 or 4] and polynucleotides closely related theretoand variants thereof.

[0047] In another particularly preferred embodiment of the inventionthere is a Histidine kinase polypeptide from Streptococcus pneumoniaecomprising or consisting of an amino acid sequence of Table 1 [SEQ IDNO: 2 or 4], or a variant thereof.

[0048] Using the information provided herein, such as a polynucleotidesequence set out in Table 1 [SEQ ID NO: 1 or 3], a polynucleotide of theinvention encoding Histidine kinase polypeptide may be obtained usingstandard cloning and screening methods, such as those for cloning andsequencing chromosomal DNA fragments from bacteria using Streptococcuspneumoniae 0100993 cells as starting material, followed by obtaining afull length clone. For example, to obtain a polynucleotide sequence ofthe invention, such as a polynucleotide sequence given in Table 1 [SEQID NO: 1 or 3], typically a library of clones of chromosomal DNA ofStreptococcus pneumoniae 0100993 in E.coli or some other suitable hostis probed with a radiolabeled oligonucleotide, preferably a 17-mer orlonger, derived from a partial sequence. Clones carrying DNA identicalto that of the probe can then be distinguished using stringenthybridization conditions. By sequencing the individual clones thusidentified by hybridization with sequencing primers designed from theoriginal polypeptide or polynucleotide sequence it is then possible toextend the polynucleotide sequence in both directions to determine afull length gene sequence. Conveniently, such sequencing is performed,for example, using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).(see in particular Screening By Hybridization 1.90 and SequencingDenatured Double-Stranded DNA Templates 13.70). Direct genomic DNAsequencing may also be performed to obtain a full length gene sequence.Illustrative of the invention, each polynucleotide set out in Table 1[SEQ ID NO: 1 or 3] was discovered in a DNA library derived fromStreptococcus pneumoniae 0100993.

[0049] Moreover, each DNA sequence set out in Table 1 [SEQ ID NO: 1 or3] contains an open reading frame encoding a protein having about thenumber of amino acid residues set forth in Table 1 [SEQ ID NO: 2 or 4]with a deduced molecular weight that can be calculated using amino acidresidue molecular weight values well known to those skilled in the art.The polynucleotide of SEQ ID NO: 1, between nucleotide number 198 andthe stop codon which begins at nucleotide number 1524 of SEQ ID NO: 1,encodes the polypeptide of SEQ ID NO: 2.

[0050] In a further aspect, the present invention provides for anisolated polynucleotide comprising or consisting of:

[0051] (a) a polynucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% or exact identity to SEQ ID NO: 1 over the entire length of SEQID NO: 1;

[0052] (b) a polynucleotide sequence encoding a polypeptide which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% or 100% exact, to the amino acid sequence ofSEQ ID NO: 2, over the entire length of SEQ ID NO: 2; or

[0053] (c) a nucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% or 100% identity, to SEQ ID NO: 1 over the entire length of SEQID NO: 3;

[0054] (d) a nucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% or exact identity to SEQ ID NO: 3 over the entire length of SEQID NO: 3; or

[0055] (e) a polynucleotide sequence encoding a polypeptide which has atleast 70% identity, preferably at least 80% identity, more preferably atleast 90% identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% or exact identity, to the amino acid sequenceof SEQ ID NO: 4, over the entire length of SEQ ID NO: 4.

[0056] A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Streptococcuspneumoniae, may be obtained by a process which comprises the steps ofscreening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO: 1 or 3 or a fragment thereof; andisolating a full-length gene and/or genomic clones containing saidpolynucleotide sequence.

[0057] The invention provides a polynucleotide sequence identical overits entire length to a coding sequence (open reading frame) in Table 1[SEQ ID NO: 1 or 3]. Also provided by the invention is a coding sequencefor a mature polypeptide or a fragment thereof, by itself as well as acoding sequence for a mature polypeptide or a fragment in reading framewith another coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also contain at least one non-codingsequence, including for example, but not limited to at least onenon-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of the fused polypeptide can be encoded.In certain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof which may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

[0058] A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 198 to the nucleotide immediatelyupstream of or including nucleotide 1524 set forth in SEQ ID NO: 1 ofTable 1, both of which encode the Histidine kinase polypeptide.

[0059] The invention also includes a polynucleotide consisting of orcomprising a polynucleotide of the formula:

X−(R ₁)_(m)−(R ₂)−(R ₃)_(n) −Y

[0060] wherein, at the 5′ end of the molecule, X is hydrogen, a metal ora modified nucleotide residue, or together with Y defines a covalentbond, and at the 3′ end of the molecule, Y is hydrogen, a metal, or amodified nucleotide residue, or together with X defines the covalentbond, each occurrence of R₁ and R₃ is independently any nucleic acidresidue or modified nucleic acid residue, m is an integer between 1 and3000 or zero , n is an integer between 1 and 3000 or zero, and R₂ is anucleic acid sequence or modified nucleic acid sequence of theinvention, particularly a nucleic acid sequence selected from Table 1 ora modified nucleic acid sequence thereof. In the polynucleotide formulaabove, R₂ is oriented so that its 5′ end nucleic acid residue is at theleft, bound to R₁, and its 3′ end nucleic acid residue is at the right,bound to R₃. Any stretch of nucleic acid residues denoted by either R₁and/or R₂, where m and/or n is greater than 1, may be either aheteropolymer or a homopolymer, preferably a heteropolymer. Where, in apreferred embodiment, X and Y together define a covalent bond, thepolynucleotide of the above formula is a closed, circularpolynucleotide, which can be a double-stranded polynucleotide whereinthe formula shows a first strand to which the second strand iscomplementary. In another preferred embodiment m and/or n is an integerbetween 1 and 1000. Other preferred embodiments of the invention areprovided where m is an integer between 1 and 50, 100 or 500, and n is aninteger between 1 and 50, 100, or 500.

[0061] It is most preferred that a polynucleotide of the invention isderived from Streptococcus pneumoniae, however, it may preferably beobtained from other organisms of the same taxonomic genus. Apolynucleotide of the invention may also be obtained, for example, fromorganisms of the same taxonomic family or order.

[0062] The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniaeHistidine kinase having an amino acid sequence set out in Table 1 [SEQID NO: 2 or 4]. The term also encompasses polynucleotides that include asingle continuous region or discontinuous regions encoding thepolypeptide (for example, polynucleotides interrupted by integratedphage, an integrated insertion sequence, an integrated vector sequence,an integrated transposon sequence, or due to RNA editing or genomic DNAreorganization) together with additional regions, that also may containcoding and/or non-coding sequences.

[0063] The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO: 2 or 4]. Fragments of apolynucleotides of the invention may be used, for example, to synthesizefull-length polynucleotides of the invention.

[0064] Further particularly preferred embodiments are polynucleotidesencoding Histidine kinase variants, that have the amino acid sequence ofHistidine kinase polypeptide of Table 1 [SEQ ID NO: 2 or 4] in whichseveral, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residuesare substituted, modified, deleted and/or added, in any combination.Especially preferred among these are silent substitutions, additions anddeletions, that do not alter the properties and activities of Histidinekinase polypeptide.

[0065] Further preferred embodiments of the invention arepolynucleotides that are at least 70% identical over their entire lengthto a polynucleotide encoding Histidine kinase polypeptide having anamino acid sequence set out in Table 1 [SEQ ID NO: 2 or 4], andpolynucleotides that are complementary to such polynucleotides.Alternatively, most highly preferred are polynucleotides that comprise aregion that is at least 80% identical over its entire length to apolynucleotide encoding Histidine kinase polypeptide and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical over their entire length to the same are particularlypreferred, and among these particularly preferred polynucleotides, thosewith at least 95% are especially preferred. Furthermore, those with atleast 97% are highly preferred among those with at least 95%, and amongthese those with at least 98% and at least 99% are particularly highlypreferred, with at least 99% being the more preferred.

[0066] Preferred embodiments are polynucleotides encoding polypeptidesthat retain substantially the same biological function or activity asthe mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO: 1 or 3].

[0067] In accordance with certain preferred embodiments of thisinvention there are provided polynucleotides that hybridize,particularly under stringent conditions, to Histidine kinasepolynucleotide sequences, such as those polynucleotides in Table 1.

[0068] The invention further relates to polynucleotides that hybridizeto the polynucleotide sequences provided herein. In this regard, theinvention especially relates to polynucleotides that hybridize understringent conditions to the polynucleotides described herein. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization occurring only if there is at least 95%and preferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

[0069] The invention also provides a polynucleotide consisting of orcomprising a polynucleotide sequence obtained by screening anappropriate library containing the complete gene for a polynucleotidesequence set forth in SEQ ID NO: 1 or 3 under stringent hybridizationconditions with a probe having the sequence of said polynucleotidesequence set forth in SEQ ID NO: 1 or 3 or a fragment thereof; andisolating said polynucleotide sequence. Fragments useful for obtainingsuch a polynucleotide include, for example, probes and primers fullydescribed elsewhere herein.

[0070] As discussed elsewhere herein regarding polynucleotide assays ofthe invention, for instance, the polynucleotides of the invention, maybe used as a hybridization probe for RNA, cDNA and genomic DNA toisolate full-length cDNAs and genomic clones encoding Histidine kinaseand to isolate cDNA and genomic clones of other genes that have a highidentity, particularly high sequence identity, to the Histidine kinasegene. Such probes generally will comprise at least 15 nucleotideresidues or base pairs. Preferably, such probes will have at least 30nucleotide residues or base pairs and may have at least 50 nucleotideresidues or base pairs. Particularly preferred probes will have at least20 nucleotide residues or base pairs and will have lee than 30nucleotide residues or base pairs.

[0071] A coding region of a Histidine kinase gene may be isolated byscreening using a DNA sequence provided in Table 1 [SEQ ID NO: 1 or 3]to synthesize an oligonucleotide probe. A labeled oligonucleotide havinga sequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

[0072] There are several methods available and well known to thoseskilled in the art to obtain full-length DNAs, or extend short DNAs, forexample those based on the method of Rapid Amplification of cDNA ends(RACE) (see, for example, Frohman, et al., PNAS USA 85: 8998-9002,1988). Recent modifications of the technique, exemplified by theMarathon™ technology (Clontech Laboratories Inc.) for example, havesignificantly simplified the search for longer cDNAs. In the Marathon™technology, cDNAs have been prepared from mRNA extracted from a chosentissue and an ‘adaptor’ sequence ligated onto each end. Nucleic acidamplification (PCR) is then carried out to amplify the “missing” 5′ endof the DNA using a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the known gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

[0073] The polynucleotides and polypeptides of the invention may beemployed, for example, as research reagents and materials for discoveryof treatments of and diagnostics for diseases, particularly humandiseases, as further discussed herein relating to polynucleotide assays.

[0074] The polynucleotides of the invention that are oligonucleotidesderived from a sequence of Table 1 [SEQ ID NOS: 1 or 2 or 3 or 4] may beused in the processes herein as described, but preferably for PCR, todetermine whether or not the polynucleotides identified herein in wholeor in part are transcribed in bacteria in infected tissue. It isrecognized that such sequences will also have utility in diagnosis ofthe stage of infection and type of infection the pathogen has attained.

[0075] The invention also provides polynucleotides that encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

[0076] For each and every polynucleotide of the invention there isprovided a polynucleotide complementary to it. It is preferred thatthese complementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

[0077] A precursor protein, having a mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0078] In addition to the standard A, G, C, T/U representations fornucleotides, the term “N” may also be used in describing certainpolynucleotides of the invention. “N” means that any of the four DNA orRNA nucleotides may appear at such a designated position in the DNA orRNA sequence, except it is preferred that N is not a nucleic acid thatwhen taken in combination with adjacent nucleotide positions, when readin the correct reading frame, would have the effect of generating apremature termination codon in such reading frame.

[0079] In sum, a polynucleotide of the invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

[0080] Vectors, Host Cells, Expression Systems

[0081] The invention also relates to vectors that comprise apolynucleotide or polynucleotides of the invention, host cells that aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

[0082] Recombinant polypeptides of the present invention may be preparedby processes well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systemswhich comprise a polynucleotide or polynucleotides of the presentinvention, to host cells which are genetically engineered with suchexpression systems, and to the production of polypeptides of theinvention by recombinant techniques.

[0083] For recombinant production of the polypeptides of the invention,host cells can be genetically engineered to incorporate expressionsystems or portions thereof or polynucleotides of the invention.Introduction of a polynucleotide into the host cell can be effected bymethods described in many standard laboratory manuals, such as Davis, etal., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calciumphosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

[0084] Representative examples of appropriate hosts include bacterialcells, such as cells of streptococci, staphylococci, enterococci E.coli, streptomyces, cyanobacteria, Bacillus subtilis, and Streptococcuspneumoniae; fingal cells, such as cells of a yeast, Kluveromyces,Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insectcells such as cells of Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanomacells; and plant cells, such as cells of a gymnosperm or angiosperm.

[0085] A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picomaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

[0086] In recombinant expression systems in eukaryotes, for secretion ofa translated protein into the lumen of the endoplasmic reticulum, intothe periplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

[0087] Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

[0088] Diagnostic, Prognostic, Serotyping and Mutation Assays

[0089] This invention is also related to the use of Histidine kinasepolynucleotides and polypeptides of the invention for use as diagnosticreagents. Detection of Histidine kinase polynucleotides and/orpolypeptides in a eukaryote, particularly a mammal, and especially ahuman, will provide a diagnostic method for diagnosis of disease,staging of disease or response of an infectious organism to drugs.Eukaryotes, particularly mammals, and especially humans, particularlythose infected or suspected to be infected with an organism comprisingthe Histidine kinase gene or protein, may be detected at the nucleicacid or amino acid level by a variety of well known techniques as wellas by methods provided herein.

[0090] Polypeptides and polynucleotides for prognosis, diagnosis orother analysis may be obtained from a putatively infected and/orinfected individual's bodily materials. Polynucleotides from any ofthese sources, particularly DNA or RNA, may be used directly fordetection or may be amplified enzymatically by using PCR or any otheramplification technique prior to analysis. RNA, particularly mRNA, cDNAand genomic DNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled Histidine kinase polynucleotidesequences. Perfectly or significantly matched sequences can bedistinguished from imperfectly or more significantly mismatched duplexesby DNase or RNase digestion, for DNA or RNA respectively, or bydetecting differences in melting temperatures or renaturation kinetics.Polynucleotide sequence differences may also be detected by alterationsin the electrophoretic mobility of polynucleotide fragments in gels ascompared to a reference sequence. This may be carried out with orwithout denaturing agents. Polynucleotide differences may also bedetected by direct DNA or RNA sequencing. See, for example, Myers etal., Science, 230: 1242 (1985). Sequence changes at specific locationsalso may be revealed by nuclease protection assays, such as RNase, V1and S1 protection assay or a chemical cleavage method. See, for example,Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).

[0091] In another embodiment, an array of oligonucleotides probescomprising Histidine kinase nucleotide sequence or fragments thereof canbe constructed to conduct efficient screening of, for example, geneticmutations, serotype, taxonomic classification or identification. Arraytechnology methods are well known and have general applicability and canbe used to address a variety of questions in molecular geneticsincluding gene expression, genetic linkage, and genetic variability(see, for example, Chee et al., Science, 274: 610 (1996)).

[0092] Thus in another aspect, the present invention relates to adiagnostic kit which comprises:

[0093] (a) a polynucleotide of the present invention, preferably thenucleotide sequence of SEQ ID NO: 1 or 3, or a fragment thereof;

[0094] (b) a nucleotide sequence complementary to that of (a);

[0095] (c) a polypeptide of the present invention, preferably thepolypeptide of SEQ ID NO: 2 or 4 or a fragment thereof; or

[0096] (d) an antibody to a polypeptide of the present invention,preferably to the polypeptide of SEQ ID NO: 2 or 4.

[0097] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

[0098] This invention also relates to the use of polynucleotides of thepresent invention as diagnostic reagents. Detection of a mutated form ofa polynucleotide of the invention, preferable, SEQ ID NO: 1 or 3, whichis associated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, which results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

[0099] The nucleotide sequences of the present invention are alsovaluable for organism chromosome identification. The sequence isspecifically targeted to, and can hybridize with, a particular locationon an organism's chromosome, particularly to a Streptococcus pneumoniaechromosome. The mapping of relevant sequences to chromosomes accordingto the present invention may be an important step in correlating thosesequences with pathogenic potential and/or an ecological niche of anorganism and/or drug resistance of an organism, as well as theessentiality of the gene to the organism. Once a sequence has beenmapped to a precise chromosomal location, the physical position of thesequence on the chromosome can be correlated with genetic map data. Suchdata may be found on-line in a sequence database. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through known genetic methods, for example,through linkage analysis (coinheritance of physically adjacent genes) ormating studies, such as by conjugation.

[0100] The differences in a polynucleotide and/or polypeptide sequencebetween organisms possessing a first phenotype and organisms possessinga different, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

[0101] Cells from an organism carrying mutations or polymorphisms(allelic variations) in a polynucleotide and/or polypeptide of theinvention may also be detected at the polynucleotide or polypeptidelevel by a variety of techniques, to allow for serotyping, for example.For example, RT-PCR can be used to detect mutations in the RNA. It isparticularly preferred to use RT-PCR in conjunction with automateddetection systems, such as, for example, GeneScan. RNA, cDNA or genomicDNA may also be used for the same purpose, PCR. As an example, PCRprimers complementary to a polynucleotide encoding Histidine kinasepolypeptide can be used to identify and analyze mutations. Examples ofrepresentative primers are shown below in Table 2. TABLE 2 Primers foramplification of Histidine kinase polynucleotides SEQ ID NO PRIMERSEQUENCE 7 5′-ATGAAACGAACAGGTTTATTTAC-3′ 85′-GTCTTGGACGACTTTTGGAAAATC-3′

[0102] The invention also includes primers of the formula:

X−(R ₁)_(m)−(R ₂)−(R ₃)_(n) −Y

[0103] wherein, at the 5′ end of the molecule, X is hydrogen, a metal ora modified nucleotide residue, and at the 3′ end of the molecule, Y ishydrogen, a metal or a modified nucleotide residue, R₁ and R₃ are anynucleic acid residue or modified nucleotide residue, m is an integerbetween 1 and 20 or zero , n is an integer between 1 and 20 or zero, andR₂ is a primer sequence of the invention, particularly a primer sequenceselected from Table 2. In the polynucleotide formula above R₂ isoriented so that its 5′ end nucleotide residue is at the left, bound toR₁, and its 3′ end nucleotide residue is at the right, bound to R₃. Anystretch of nucleic acid residues denoted by either R group, where mand/or n is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer being complementary to a regionof a polynucleotide of Table 1. In a preferred embodiment m and/or n isan integer between 1 and 10.

[0104] The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5′ and/or the 3′ end. These primers may beused for, among other things, amplifying Histidine kinase DNA and/or RNAisolated from a sample derived from an individual, such as a bodilymaterial. The primers may be used to amplify a polynucleotide isolatedfrom an infected individual, such that the polynucleotide may then besubject to various techniques for elucidation of the polynucleotidesequence. In this way, mutations in the polynucleotide sequence may bedetected and used to diagnose and/or prognose the infection or its stageor course, or to serotype and/or classify the infectious agent.

[0105] The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byStreptococcus pneumoniae, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO: 1or 3]. Increased or decreased expression of a Histidine kinasepolynucleotide can be measured using any on of the methods well known inthe art for the quantitation of polynucleotides, such as, for example,amplification, PCR, RT-PCR, RNase protection, Northern blotting,spectrometry and other hybridization methods.

[0106] In addition, a diagnostic assay in accordance with the inventionfor detecting over-expression of Histidine kinase polypeptide comparedto normal control tissue samples may be used to detect the presence ofan infection, for example. Assay techniques that can be used todetermine levels of a Histidine kinase polypeptide, in a sample derivedfrom a host, such as a bodily material, are well-known to those of skillin the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis, antibody sandwichassays, antibody detection and ELISA assays.

[0107] Differential Expression

[0108] The polynucleotides and polynucleotides of the invention may beused as reagents for differential screening methods. There are manydifferential screening and differential display methods known in the artin which the polynucleotides and polypeptides of the invention may beused. For example, the differential display technique is described byChuang et al., J. Bacteriol. 175:2026-2036 (1993). This methodidentifies those genes which are expressed in an organism by identifyingmRNA present using randomly-primed RT-PCR. By comparing pre-infectionand post infection profiles, genes up and down regulated duringinfection can be identified and the RT-PCR product sequenced and matchedto ORF “unknowns.”

[0109] In Vivo Expression Technology (IVET) is described by Camilli etal., Proc. Nat'l Acad. Sci. USA. 91:2634-2638 (1994). IVET identifiesgenes up-regulated during infection when compared to laboratorycultivation, implying an important role in infection. ORFs identified bythis technique are implied to have a significant role in infectionestablishment and/or maintenance. In this technique random chromosomalfragments of target organism are cloned upstream of a promoter-lessrecombinase gene in a plasmid vector. This construct is introduced intothe target organism which carries an antibiotic resistance gene flankedby resolvase sites. Growth in the presence of the antibiotic removesfrom the population those fragments cloned into the plasmid vectorcapable of supporting transcription of the recombinase gene andtherefore have caused loss of antibiotic resistance. The resistant poolis introduced into a host and at various times after infection bacteriamay be recovered and assessed for the presence of antibiotic resistance.The chromosomal fragment carried by each antibiotic sensitive bacteriumshould carry a promoter or portion of a gene normally upregulated duringinfection. Sequencing upstream of the recombinase gene allowsidentification of the up regulated gene.

[0110] RT-PCR may also be used to analyze gene expression patterns. ForRT PCR using the polynucleotides of the invention, messenger RNA isisolated from bacterial infected tissue, e.g., 48 hour murine lunginfections, and the amount of each mRNA species assessed by reversetranscription of the RNA sample primed with random hexanucleotidesfollowed by PCR with gene specific primer pairs. The determination ofthe presence and amount of a particular mRNA species by quantificationof the resultant PCR product provides information on the bacterial geneswhich are transcribed in the infected tissue. Analysis of genetranscription can be carried out at different times of infection to gaina detailed knowledge of gene regulation in bacterial pathogenesisallowing for a clearer understanding of which gene products representtargets for screens for antibacterials. Because of the gene specificnature of the PCR primers employed it should be understood that thebacterial mRNA preparation need not be free of mammalian RNA. Thisallows the investigator to carry out a simple and quick RNA preparationfrom infected tissue to obtain bacterial mRNA species which are veryshort lived in the bacterium (in the order of 2 minute halflives).Optimally the bacterial mRNA is prepared from infected murine lungtissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL)for very short periods of time, subsequent processing according to themanufacturers of TRIzole reagent and DNAase treatment to removecontaminating DNA. Preferably the process is optimized by finding thoseconditions which give a maximum amount of Streptococcus pneumoniae 16Sribosomal RNA as detected by probing Northerns with a suitably labeledsequence specific oligonucleotide probe. Typically a 5′ dye labeledprimer is used in each PCR primer pair in a PCR reaction which isterminated optimally between 8 and 25 cycles. The PCR products areseparated on 6% polyacrylamide gels with detection and quantificationusing GeneScanner (manufactured by ABI).

[0111] Gridding and Polynucleotide Subtraction

[0112] Methods have been described for obtaining information about geneexpression and identity using so called “high density DNA arrays” orgrids. See, e.g., M. Chee et al., Science, 274:610-614 (1996) and otherreferences cited therein. Such gridding assays have been employed toidentify certain novel gene sequences, referred to as Expressed SequenceTags (EST) (Adams et a., Science, 252:1651-1656 (1991)). A variety oftechniques have also been described for identifying particular genesequences on the basis of their gene products. For example, seeInternational Patent Application No. WO91/07087, published May 30, 1991.In addition, methods have been described for the amplification ofdesired sequences. For example, see International Patent Application No.WO91/17271, published Nov. 14, 1991.

[0113] The polynucleotides of the invention may be used as components ofpolynucleotide arrays, preferably high density arrays or grids. Thesehigh density arrays are particularly useful for diagnostic andprognostic purposes. For example, a set of spots each comprising adifferent gene, and further comprising a polynucleotide orpolynucleotides of the invention, may be used for probing, such as usinghybridization or nucleic acid amplification, using a probes obtained orderived from a bodily sample, to determine the presence of a particularpolynucleotide sequence or related sequence in an individual. Such apresence may indicate the presence of a pathogen, particularlyStreptococcus pneumoniae, and may be useful in diagnosing and/orprognosing disease or a course of disease. A grid comprising a number ofvariants of the polynucleotide sequence of SEQ ID NO: 1 or 3 arepreferred. Also preferred is a comprising a number of variants of apolynucleotide sequence encoding the polypeptide sequence of SEQ ID NO:2 or 4.

[0114] Antibodies

[0115] The polypeptides and polynucleotides of the invention or variantsthereof, or cells expressing the same can be used as immunogens toproduce antibodies immunospecific for such polypeptides orpolynucleotides respectively.

[0116] In certain preferred embodiments of the invention there areprovided antibodies against Histidine kinase polypeptides orpolynucleotides.

[0117] Antibodies generated against the polypeptides or polynucleotidesof the invention can be obtained by administering the polypeptidesand/or polynucleotides of the invention, or epitope-bearing fragments ofeither or both, analogues of either or both, or cells expressing eitheror both, to an animal, preferably a nonhuman, using routine protocols.For preparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0118] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies topolypeptides or polynucleotides of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies immunospecific to the polypeptides orpolynucleotides of the invention.

[0119] Alternatively, phage display technology may be utilized to selectantibody genes with binding activities towards a polypeptide of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-Histidine kinase orfrom naive libraries (McCafferty, et aL, (1990), Nature 348, 552-554;Marks, et al., (1992) Biotechnology 10, 779-783). The affinity of theseantibodies can also be improved by, for example, chain shuffling(Clackson et al, (1991) Nature 352: 628).

[0120] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptides or polynucleotides of theinvention to purify the polypeptides or polynucleotides by, for example,affinity chromatography.

[0121] Thus, among others, antibodies against Histidinekinase-polypeptide or Histidine kinase-polynucleotide may be employed totreat infections, particularly bacterial infections.

[0122] Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants form a particular aspect of thisinvention.

[0123] A polypeptide or polynucleotide of the invention, such as anantigenically or immunologically equivalent derivative or a fusionprotein of the polypeptide is used as an antigen to immunize a mouse orother animal such as a rat or chicken. The fusion protein may providestability to the polypeptide. The antigen may be associated, for exampleby conjugation, with an immunogenic carrier protein for example bovineserum albumin, keyhole limpet haemocyanin or tetanus toxoid.Alternatively, a multiple antigenic polypeptide comprising multiplecopies of the polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

[0124] Preferably, the antibody or variant thereof is modified to makeit less immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized,” where thecomplimentarity determining region or regions of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones et al. (1986), Nature 321, 522-525 orTempest et al., (1991) Biotechnology 9, 266-273.

[0125] In accordance with an aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of Histidine kinase polynucleotides andpolypeptides encoded thereby.

[0126] The use of a polynucleotide of the invention in geneticimmunization will preferably employ a suitable delivery method such asdirect injection of plasmid DNA into muscles (Wolff et al., Hum MolGenet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419),delivery of DNA complexed with specific protein carriers (Wu et al., JBiol Chem. (1989) 264: 16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS USA, (1986) 83: 9551),encapsulation of DNA in various forms of liposomes (Kaneda et al.,Science (1989) 243: 375), particle bombardment (Tang et al., Nature(1992) 356:152, Eisenbraun et al., DNA Cell Biol (1993) 12: 791) and invivo infection using cloned retroviral vectors (Seeger et al., PNAS USA(1984) 81: 5849).

[0127] Antagonists and Agonists—Assays and Molecules

[0128] Polypeptides and polynucleotides of the invention may also beused to assess the binding of small molecule substrates and ligands in,for example, cells, cell-free preparations, chemical libraries, andnatural product mixtures. These substrates and ligands may be naturalsubstrates and ligands or may be structural or functional mimetics. See,e.g., Coligan et al., Current Protocols in Immunology 1(2): Chapter 5(1991).

[0129] Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular the Diseases hereinbefore mentioned. It istherefore desirable to devise screening methods to identify compoundswhich stimulate or which inhibit the function of the polypeptide orpolynucleotide. Accordingly, in a further aspect, the present inventionprovides for a method of screening compounds to identify those whichstimulate or which inhibit the function of a polypeptide orpolynucleotide of the invention, as well as related polypeptides andpolynucleotides. In general, agonists or antagonists may be employed fortherapeutic and prophylactic purposes for such Diseases as hereinbeforementioned. Compounds may be identified from a variety of sources, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. Such agonists, antagonists or inhibitors so-identifiedmay be natural or modified substrates, ligands, receptors, enzymes,etc., as the case may be, of Histidine kinase polypeptides andpolynucleotides; or may be structural or functional mimetics thereof(see Coligan et al., Current Protocols in Immunology 1(2):Chapter 5(1991)).

[0130] The screening methods may simply measure the binding of acandidate compound to the polypeptide or polynucleotide, or to cells ormembranes bearing the polypeptide or polynucleotide, or a fusion proteinof the polypeptide by means of a label directly or indirectly associatedwith the candidate compound. Alternatively, the screening method mayinvolve competition with a labeled competitor. Further, these screeningmethods may test whether the candidate compound results in a signalgenerated by activation or inhibition of the polypeptide orpolynucleotide, using detection systems appropriate to the cellscomprising the polypeptide or polynucleotide. Inhibitors of activationare generally assayed in the presence of a known agonist and the effecton activation by the agonist by the presence of the candidate compoundis observed. Constitutively active polypeptide and/or constitutivelyexpressed polypeptides and polynucleotides may be employed in screeningmethods for inverse agonists or inhibitors, in the absence of an agonistor inhibitor, by testing whether the candidate compound results ininhibition of activation of the polypeptide or polynucleotide, as thecase may be. Further, the screening methods may simply comprise thesteps of mixing a candidate compound with a solution containing apolypeptide or polynucleotide of the present invention, to form amixture, measuring Histidine kinase polypeptide and/or polynucleotideactivity in the mixture, and comparing the Histidine kinase polypeptideand/or polynucleotide activity of the mixture to a standard. Fusionproteins, such as those made from Fc portion and Histidine kinasepolypeptide, as hereinbefore described, can also be used forhigh-throughput screening assays to identify antagonists of thepolypeptide of the present invention, as well as of phylogenetically andand/or functionally related polypeptides (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

[0131] The polynucleotides, polypeptides and antibodies that bind toand/or interact with a polypeptide of the present invention may also beused to configure screening methods for detecting the effect of addedcompounds on the production of mRNA and/or polypeptide in cells. Forexample, an ELISA assay may be constructed for measuring secreted orcell associated levels of polypeptide using monoclonal and polyclonalantibodies by standard methods known in the art. This can be used todiscover agents which may inhibit or enhance the production ofpolypeptide (also called antagonist or agonist, respectively) fromsuitably manipulated cells or tissues.

[0132] The invention also provides a method of screening compounds toidentify those which enhance (agonist) or block (antagonist) the actionof Histidine kinase polypeptides or polynucleotides, particularly thosecompounds that are bacteristatic and/or bactericidal. The method ofscreening may involve high-throughput techniques. For example, to screenfor agonists or antagonists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising Histidine kinase polypeptide anda labeled substrate or ligand of such polypeptide is incubated in theabsence or the presence of a candidate molecule that may be a Histidinekinase agonist or antagonist. The ability of the candidate molecule toagonize or antagonize the Histidine kinase polypeptide is reflected indecreased binding of the labeled ligand or decreased production ofproduct from such substrate. Molecules that bind gratuitously, i.e.,without inducing the effects of Histidine kinase polypeptide are mostlikely to be good antagonists. Molecules that bind well and, as the casemay be, increase the rate of product production from substrate, increasesignal transduction, or increase chemical channel activity are agonists.Detection of the rate or level of, as the case may be, production ofproduct from substrate, signal transduction, or chemical channelactivity may be enhanced by using a reporter system. Reporter systemsthat may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in Histidine kinase polynucleotide orpolypeptide activity, and binding assays known in the art. Polypeptidesof the invention may be used to identify membrane bound or solublereceptors, if any, for such polypeptide, through standard receptorbinding techniques known in the art. These techniques include, but arenot limited to, ligand binding and crosslinking assays in which thepolypeptide is labeled with a radioactive isotope (for instance, ¹²⁵I),chemically modified (for instance, biotinylated), or fused to a peptidesequence suitable for detection or purification, and incubated with asource of the putative receptor (e.g., cells, cell membranes, cellsupernatants, tissue extracts, bodily materials). Other methods includebiophysical techniques such as surface plasmon resonance andspectroscopy. These screening methods may also be used to identifyagonists and antagonists of the polypeptide which compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

[0133] The fluorescence polarization value for a fluorescently-taggedmolecule depends on the rotational correlation time or tumbling rate.Protein complexes, such as formed by Histidine kinase polypeptideassociating with another Histidine kinase polypeptide or otherpolypeptide, labeled to comprise a fluorescently-labeled molecule willhave higher polarization values than a fluorescently labeled monomericprotein. It is preferred that this method be used to characterize smallmolecules that disrupt polypeptide complexes.

[0134] Fluorescence energy transfer may also be used characterize smallmolecules that interfere with the formation of Histidine kinasepolypeptide dimers, trimers, tetramers or higher order structures, orstructures formed by Histidine kinase polypeptide bound to anotherpolypeptide. Histidine kinase polypeptide can be labeled with both adonor and acceptor fluorophore. Upon mixing of the two labeled speciesand excitation of the donor fluorophore, fluorescence energy transfercan be detected by observing fluorescence of the acceptor. Compoundsthat block dimerization will inhibit fluorescence energy transfer.

[0135] Surface plasmon resonance can be used to monitor the effect ofsmall molecules on Histidine kinase polypeptide self-association as wellas an association of Histidine kinase polypeptide and anotherpolypeptide or small molecule. Histidine kinase polypeptide can becoupled to a sensor chip at low site density such that covalently boundmolecules will be monomeric. Solution protein can then passed over theHistidine kinase polypeptide-coated surface and specific binding can bedetected in real-time by monitoring the change in resonance angle causedby a change in local refractive index. This technique can be used tocharacterize the effect of small molecules on kinetic rates andequilibrium binding constants for Histidine kinase polypeptideself-association as well as an association of Histidine kinasepolypeptide and another polypeptide or small molecule.

[0136] A scintillation proximity assay may be used to characterize theinteraction between an association of Histidine kinase polypeptide withanother Histidine kinase polypeptide or a different polypeptide.Histidine kinase polypeptide can be coupled to a scintillation-filledbead. Addition of radio-labeled Histidine kinase polypeptide results inbinding where the radioactive source molecule is in close proximity tothe scintillation fluid. Thus, signal is emitted upon Histidine kinasepolypeptide binding and compounds that prevent Histidine kinasepolypeptide self-association or an association of Histidine kinasepolypeptide and another polypeptide or small molecule will diminishsignal.

[0137] ICS biosensors have been described by AMBRI (Australian MembraneBiotechnology Research Institute). They couple the self-association ofmacromolecules to the closing of gramacidin-facilitated ion channels insuspended membrane bilayers and hence to a measurable change in theadmittance (similar to impedence) of the biosensor. This approach islinear over six decades of admittance change and is ideally suited forlarge scale, high through-put screening of small molecule combinatoriallibraries.

[0138] In other embodiments of the invention there are provided methodsfor identifying compounds which bind to or otherwise interact with andinhibit or activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contacting a polypeptideand/or polynucleotide of the invention with a compound to be screenedunder conditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detectable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound; and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

[0139] Another example of an assay for Histidine kinase agonists is acompetitive assay that combines Histidine kinase and a potential agonistwith Histidine kinase-binding molecules, recombinant Histidine kinasebinding molecules, natural substrates or ligands, or substrate or ligandmimetics, under appropriate conditions for a competitive inhibitionassay. Histidine kinase can be labeled, such as by radioactivity or acolorimetric compound, such that the number of Histidine kinasemolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

[0140] Potential antagonists include, among others, small organicmolecules, peptides, polypeptides and antibodies that bind to apolynucleotide and/or polypeptide of the invention and thereby inhibitor extinguish its activity or expression. Potential antagonists also maybe small organic molecules, a peptide, a polypeptide such as a closelyrelated protein or antibody that binds the same sites on a bindingmolecule, such as a binding molecule, without inducing Histidinekinase-induced activities, thereby preventing the action or expressionof Histidine kinase polypeptides and/or polynucleotides by excludingHistidine kinase polypeptides and/or polynucleotides from binding.

[0141] Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of Histidine kinase.

[0142] Other examples of potential polypeptide antagonists includeantibodies or, in some cases, oligonucleotides or proteins which areclosely related to the ligands, substrates, receptors, enzymes, etc., asthe case may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or small molecules which bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented.

[0143] Certain of the polypeptides of the invention are biomimetics,functional mimetics of the natural Histidine kinase polypeptide. Thesefunctional mimetics may be used for, among other things, antagonizingthe activity of Histidine kinase polypeptide or as a antigen orimmunogen in a manner described elsewhere herein. Functional mimetics ofthe polypeptides of the invention include but are not limited totruncated polypeptides. For example, preferred functional mimeticsinclude, a polypeptide comprising the polypeptide sequence set forth inSEQ ID NO: 2 lacking 20, 30, 40, 50, 60, 70 or 80 amino- orcarboxy-terminal amino acid residues, including fusion proteinscomprising one or more of these truncated sequences. Polynucleotidesencoding each of these functional mimetics may be used as expressioncassettes to express each mimetic polypeptide. It is preferred thatthese cassettes comprise 5′ and 3′ restriction sites to allow for aconvenient means to ligate the cassettes together when desired. It isfurther preferred that these cassettes comprise gene expression signalsknown in the art or described elsewhere herein.

[0144] Thus, in another aspect, the present invention relates to ascreening kit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for a polypeptide and/or polynucleotide of thepresent invention; or compounds which decrease or enhance the productionof such polypeptides and/or polynucleotides , which comprises:

[0145] (a) a polypeptide and/or a polynucleotide of the presentinvention;

[0146] (b) a recombinant cell expressing a polypeptide and/orpolynucleotide of the present invention;

[0147] (c) a cell membrane expressing a polypeptide and/orpolynucleotide of the present invention; or

[0148] (d) antibody to a polypeptide and/or polynucleotide of thepresent invention;

[0149] which polypeptide is preferably that of SEQ ID NO: 2, and whichpolynucleotide is preferably that of SEQ ID NO: 1.

[0150] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component.

[0151] It will be readily appreciated by the skilled artisan that apolypeptide and/or polynucleotide of the present invention may also beused in a method for the structure-based design of an agonist,antagonist or inhibitor of the polypeptide and/or polynucleotide, by:

[0152] (a) determining in the first instance the three-dimensionalstructure of the polypeptide and/or polynucleotide, or complexesthereof;

[0153] (b) deducing the three-dimensional structure for the likelyreactive site(s), binding site(s) or motif(s) of an agonist, antagonistor inhibitor;

[0154] (c) synthesizing candidate compounds that are predicted to bindto or react with the deduced binding site(s), reactive site(s), and/ormotif(s); and

[0155] (d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

[0156] It will be further appreciated that this will normally be aniterative process, and this iterative process may be performed usingautomated and computer-controlled steps.

[0157] In a further aspect, the present invention provides methods oftreating abnormal conditions such as, for instance, a Disease, relatedto either an excess of, an under-expression of, an elevated activity of,or a decreased activity of Histidine kinase polypeptide and/orpolynucleotide.

[0158] If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises administering to an individual in need thereof aninhibitor compound (antagonist) as herein described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function and/or expression of the polypeptideand/or polynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of the Histidinekinase polypeptide and/or polypeptide.

[0159] In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

[0160] In still another approach, expression of the gene encodingendogenous Histidine kinase polypeptide can be inhibited usingexpression blocking techniques. This blocking may be targeted againstany step in gene expression, but is preferably targeted againsttranscription and/or translation. An examples of a known technique ofthis sort involve the use of antisense sequences, either internallygenerated or separately administered (see, for example, O'Connor, JNeurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Alternatively,oligonucleotides which form triple helices with the gene can be supplied(see, for example, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooneyet al., Science (1988) 241:456; Dervan et al., Science (1991) 251:1360).These oligomers can be administered per se or the relevant oligomers canbe expressed in vivo.

[0161] Each of the polynucleotide sequences provided herein may be usedin the discovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

[0162] The invention also provides the use of the polypeptide,polynucleotide, agonist or antagonist of the invention to interfere withthe initial physical interaction between a pathogen or pathogens and aeukaryotic, preferably mammalian, host responsible for sequelae ofinfection. In particular, the molecules of the invention may be used: inthe prevention of adhesion of bacteria, in particular gram positiveand/or gram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block Histidine kinase protein-mediatedmammalian cell invasion by, for example, initiating phosphorylation ofmammalian tyrosine kinases (Rosenshine et al., Infect. Immun. 60:2211(1992); to block bacterial adhesion between eukaryotic, preferablymammalian, extracellular matrix proteins and bacterial Histidine kinaseproteins that mediate tissue damage and/or; to block the normalprogression of pathogenesis in infections initiated other than by theimplantation of in-dwelling devices or by other surgical techniques.

[0163] This invention provides a method of screening drugs to identifythose which interfere with i) the interaction of the histidine kinasewith a response regulator, the method comprising incubating thehistidine kinase with response regulator in the presence of the drug andmeasuring the ability of the drug to block this interaction; and/or ii)the ability of the histidine kinase to autophosphorylate, the methodcomprising incubating the histidine kinase with the drug and measuringthe ability of the drug to prevent autophosphorylation.

[0164] The response regulator is preferably the cognate responseregulator of the histidine kinase, or another response regulator whichis capable of using the histidine kinase as a substrate, and ispreferably from Streptococcus pneumoniae or another microorganism e.g.Bacillus. Polypeptide and polynucleotide sequences of the cognateresponse regulator of the Histidine kinase of the invention are setforth in Table 1 (E and F). This novel response regulator shows 36%identity to PhoP from Bacillus subtilis.

[0165] In accordance with yet another aspect of the invention, there areprovided Histidine kinase agonists and antagonists, preferablybacteristatic or bactericidal agonists and antagonists.

[0166] The antagonists and agonists of the invention may be employed,for instance, to prevent, inhibit and/or treat diseases.

[0167]Helicobacter pylori (herein “H. pylori”) bacteria infect thestomachs of over one-third of the world's population causing stomachcancer, ulcers, and gastritis (International Agency for Research onCancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori(International Agency for Research on Cancer, Lyon, France,http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofHistidine kinase polypeptides and/or polynucleotides) found usingscreens provided by the invention, or known in the art, particularlynarrow-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also prevent, inhibit and/or cure gastric ulcers andgastritis.

[0168] Vaccines

[0169] There are provided by the invention, products, compositions andmethods for assessing Histidine kinase expression, treating disease,assaying genetic variation, and administering a Histidine kinasepolypeptide and/or polynucleotide to an organism to raise animmunological response against a bacteria, especially a Streptococcuspneumoniae bacteria.

[0170] Another aspect of the invention relates to a method for inducingan immunological response in an individual, particularly a mammal whichcomprises inoculating the individual with Histidine kinasepolynucleotide and/or polypeptide, or a fragment or variant thereof,adequate to produce antibody and/or T cell immune response to protectsaid individual from infection, particularly bacterial infection andmost particularly Streptococcus pneumoniae infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector, sequence or ribozyme to directexpression of Histidine kinase polynucleotide and/or polypeptide, or afragment or a variant thereof, for expressing Histidine kinasepolynucleotide and/or polypeptide, or a fragment or a variant thereof invivo in order to induce an immunological response, such as, to produceantibody and/or T cell immune response, including, for example,cytokine-producing T cells or cytotoxic T cells, to protect saidindividual, preferably a human, from disease, whether that disease isalready established within the individual or not. One example ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, aDNA-protein complex or an RNA-protein complex.

[0171] A further aspect of the invention relates to an immunologicalcomposition that when introduced into an individual, preferably a human,capable of having induced within it an immunological response, inducesan immunological response in such individual to a Histidine kinasepolynucleotide and/or polypeptide encoded therefrom, wherein thecomposition comprises a recombinant Histidine kinase polynucleotideand/or polypeptide encoded therefrom and/or comprises DNA and/or RNAwhich encodes and expresses an antigen of said Histidine kinasepolynucleotide, polypeptide encoded therefrom, or other polypeptide ofthe invention. The immunological response may be used therapeutically orprophylactically and may take the form of antibody immunity and/orcellular immunity, such as cellular immunity arising from CTL or CD4+Tcells.

[0172] A Histidine kinase polypeptide or a fragment thereof may be fusedwith co-protein or chemical moiety which may or may not by itselfproduce antibodies, but which is capable of stabilizing the firstprotein and producing a fused or modified protein which will haveantigenic and/or immunogenic properties, and preferably protectiveproperties. Thus fused recombinant protein, preferably further comprisesan antigenic co-protein, such as lipoprotein D from Hemophilusinfluenzae, Glutathione-S-transferase (GST) or beta-galactosidase, orany other relatively large co-protein which solubilizes the protein andfacilitates production and purification thereof. Moreover, theco-protein may act as an adjuvant in the sense of providing ageneralized stimulation of the immune system of the organism receivingthe protein. The co-protein may be attached to either the amino- orcarboxy-terminus of the first protein.

[0173] Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides and/orpolynucleotides of the invention and immunostimulatory DNA sequences,such as those described in Sato, Y. et al. Science 273: 352 (1996).

[0174] Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof, which have been shown toencode non-variable regions of bacterial cell surface proteins, inpolynucleotide constructs used in such genetic immunization experimentsin animal models of infection with Streptococcus pneumoniae. Suchexperiments will be particularly useful for identifying protein epitopesable to provoke a prophylactic or therapeutic immune response. It isbelieved that this approach will allow for the subsequent preparation ofmonoclonal antibodies of particular value, derived from the requisiteorgan of the animal successfully resisting or clearing infection, forthe development of prophylactic agents or therapeutic treatments ofbacterial infection, particularly Streptococcus pneumoniae infection, inmammals, particularly humans.

[0175] A polypeptide of the invention may be used as an antigen forvaccination of a host to produce specific antibodies which protectagainst invasion of bacteria, for example by blocking adherence ofbacteria to damaged tissue. Examples of tissue damage include wounds inskin or connective tissue caused, for example, by mechanical, chemical,thermal or radiation damage or by implantation of indwelling devices, orwounds in the mucous membranes, such as the mouth, throat, mammaryglands, urethra or vagina.

[0176] The invention also includes a vaccine formulation which comprisesan immunogenic recombinant polypeptide and/or polynucleotide of theinvention together with a suitable carrier, such as a pharmaceuticallyacceptable carrier. Since the polypeptides and polynucleotides may bebroken down in the stomach, each is preferably administeredparenterally, including, for example, administration that issubcutaneous, intramuscular, intravenous, or intradermal. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteristatic compounds and solutes which render the formulationisotonic with the bodily fluid, preferably the blood, of the individual;and aqueous and non-aqueous sterile suspensions which may includesuspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

[0177] While the invention has been described with reference to certainHistidine kinase polypeptides and polynucleotides, it is to beunderstood that this covers fragments of the naturally occurringpolypeptides and polynucleotides, and similar polypeptides andpolynucleotides with additions, deletions or substitutions which do notsubstantially affect the immunogenic properties of the recombinantpolypeptides or polynucleotides.

[0178] Compositions, Kits and Administration

[0179] In a further aspect of the invention there are providedcompositions comprising a Histidine kinase polynucleotide and/or aHistidine kinase polypeptide for administration to a cell or to amulticellular organism.

[0180] The invention also relates to compositions comprising apolynucleotide and/or a polypeptides discussed herein or their agonistsor antagonists. The polypeptides and polynucleotides of the inventionmay be employed in combination with a non-sterile or sterile carrier orcarriers for use with cells, tissues or organisms, such as apharmaceutical carrier suitable for administration to an individual.Such compositions comprise, for instance, a media additive or atherapeutically effective amount of a polypeptide and/or polynucleotideof the invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

[0181] Polypeptides, polynucleotides and other compounds of theinvention may be employed alone or in conjunction with other compounds,such as therapeutic compounds.

[0182] The pharmaceutical compositions may be administered in anyeffective, convenient manner including, for instance, administration bytopical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes amongothers.

[0183] In therapy or as a prophylactic, the active agent may beadministered to an individual as an injectable composition, for exampleas a sterile aqueous dispersion, preferably isotonic.

[0184] Alternatively the composition may be formulated for topicalapplication for example in the form of ointments, creams, lotions, eyeointments, eye drops, ear drops, mouthwash, impregnated dressings andsutures and aerosols, and may contain appropriate conventionaladditives, including, for example, preservatives, solvents to assistdrug penetration, and emollients in ointments and creams. Such topicalformulations may also contain compatible conventional carriers, forexample cream or ointment bases, and ethanol or oleyl alcohol forlotions. Such carriers may constitute from about 1% to about 98% byweight of the formulation; more usually they will constitute up to about80% by weight of the formulation.

[0185] In a further aspect, the present invention provides forpharmaceutical compositions comprising a therapeutically effectiveamount of a polypeptide and/or polynucleotide, such as the soluble formof a polypeptide and/or polynucleotide of the present invention, agonistor antagonist peptide or small molecule compound, in combination with apharmaceutically acceptable carrier or excipient. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The invention furtherrelates to pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides,polynucleotides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0186] The composition will be adapted to the route of administration,for instance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, and the like.

[0187] For administration to mammals, and particularly humans, it isexpected that the daily dosage level of the active agent will be from0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in anyevent will determine the actual dosage which will be most suitable foran individual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

[0188] In-dwelling devices include surgical implants, prosthetic devicesand catheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

[0189] The composition of the invention may be administered by injectionto achieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

[0190] Many orthopedic surgeons consider that humans with prostheticjoints should be considered for antibiotic prophylaxis before dentaltreatment that could produce a bacteremia. Late deep infection is aserious complication sometimes leading to loss of the prosthetic jointand is accompanied by significant morbidity and mortality. It maytherefore be possible to extend the use of the active agent as areplacement for prophylactic antibiotics in this situation.

[0191] In addition to the therapy described above, the compositions ofthis invention may be used generally as a wound treatment agent toprevent adhesion of bacteria to matrix proteins exposed in wound tissueand for prophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

[0192] Alternatively, the composition of the invention may be used tobathe an indwelling device immediately before insertion. The activeagent will preferably be present at a concentration of 1 μg/ml to 10mg/ml for bathing of wounds or indwelling devices.

[0193] A vaccine composition is conveniently in injectable form.Conventional adjuvants may be employed to enhance the immune response. Asuitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, andsuch dose is preferably administered 1-3 times and with an interval of1-3 weeks. With the indicated dose range, no adverse toxicologicaleffects will be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

[0194] Sequence Databases, Sequences in a Tangible Medium, andAlgorithms

[0195] Polynucleotide and polypeptide sequences form a valuableinformation resource with which to determine their 2- and 3-dimensionalstructures as well as to identify further sequences of similar homology.These approaches are most easily facilitated by storing the sequence ina computer readable medium and then using the stored data in a knownmacromolecular structure program or to search a sequence database usingwell known searching tools, such as GCC.

[0196] The polynucleotide and polypeptide sequences of the invention areparticularly useful as components in databases useful for searchanalyses as well as in sequence analysis algorithms. As used in thissection entitled “Sequence Databases, Sequences in a Tangible Medium,and Algorithms,” and in claims related to this section, the terms“polynucleotide of the invention” and “polynucleotide sequence of theinvention” mean any detectable chemical or physical characteristic of apolynucleotide of the invention that is or may be reduced to or storedin a tangible medium, preferably a computer readable form. For example,chromatographic scan data or peak data, photographic data or scan datatherefrom, called bases, and mass spectrographic data. As used in thissection entitled Databases and Algorithms and in claims related thereto,the terms “polypeptide of the invention” and “polypeptide sequence ofthe invention” mean any detectable chemical or physical characteristicof a polypeptide of the invention that is or may be reduced to or storedin a tangible medium, preferably a computer readable form. For example,chromatographic scan data or peak data, photographic data or scan datatherefrom, and mass spectrographic data.

[0197] The invention provides a computer readable medium having storedthereon polypeptide sequences of the invention and/or polynucleotidesequences of the invention. For example, a computer readable medium isprovided comprising and having stored thereon a member selected from thegroup consisting of: a polynucleotide comprising the sequence of apolynucleotide of the invention; a polypeptide comprising the sequenceof a polypeptide sequence of the invention; a set of polynucleotidesequences wherein at least one of the sequences comprises the sequenceof a polynucleotide sequence of the invention; a set of polypeptidesequences wherein at least one of the sequences comprises the sequenceof a polypeptide sequence of the invention; a data set representing apolynucleotide sequence comprising the sequence of polynucleotidesequence of the invention; a data set representing a polynucleotidesequence encoding a polypeptide sequence comprising the sequence of apolypeptide sequence of the invention; a polynucleotide comprising thesequence of a polynucleotide sequence of the invention; a polypeptidecomprising the sequence of a polypeptide sequence of the invention; aset of polynucleotide sequences wherein at least one of the sequencescomprises the sequence of a polynucleotide sequence of the invention; aset of polypeptide sequences wherein at least one of said sequencescomprises the sequence of a polypeptide sequence of the invention; adata set representing a polynucleotide sequence comprising the sequenceof a polynucleotide sequence of the invention; a data set representing apolynucleotide sequence encoding a polypeptide sequence comprising thesequence of a polypeptide sequence of the invention. The computerreadable medium can be any composition of matter used to storeinformation or data, including, for example, commercially availablefloppy disks, tapes, chips, hard drives, compact disks, and video disks.

[0198] Also provided by the invention are methods for the analysis ofcharacter sequences or strings, particularly genetic sequences orencoded genetic sequences. Preferred methods of sequence analysisinclude, for example, methods of sequence homology analysis, such asidentity and similarity analysis, RNA structure analysis, sequenceassembly, cladistic analysis, sequence motif analysis, open readingframe determination, nucleic acid base calling, nucleic acid basetrimming, and sequencing chromatogram peak analysis.

[0199] A computer based method is provided for performing homologyidentification. This method comprises the steps of providing apolynucleotide sequence comprising the sequence a polynucleotide of theinvention in a computer readable medium; and comparing saidpolynucleotide sequence to at least one polynucleotide or polypeptidesequence to identify homology.

[0200] A computer based method is also provided for performing homologyidentification, said method comprising the steps of: providing apolypeptide sequence comprising the sequence of a polypeptide of theinvention in a computer readable medium; and comparing said polypeptidesequence to at least one polynucleotide or polypeptide sequence toidentify homology.

[0201] A computer based method is still further provided forpolynucleotide assembly, said method comprising the steps of: providinga first polynucleotide sequence comprising the sequence of apolynucleotide of the invention in a computer readable medium; andscreening for at least one overlapping region between said firstpolynucleotide sequence and a second polynucleotide sequence.

[0202] A further embodiment of the invention provides a computer basedmethod for performing homology identification, said method comprisingthe steps of: providing a polynucleotide sequence comprising thesequence of a polynucleotide of the invention in a computer readablemedium; and comparing said polynucleotide sequence to at least onepolynucleotide or polypeptide sequence to identify homology.

[0203] A further embodiment of the invention provides a computer basedmethod for performing homology identification, said method comprisingthe steps of: providing a polypeptide sequence comprising the sequenceof a polypeptide of the invention in a computer readable medium; andcomparing said polypeptide sequence to at least one polynucleotide orpolypeptide sequence to identify homology.

[0204] A further embodiment of the invention provides a computer basedmethod for polynucleotide assembly, said method comprising the steps of:providing a first polynucleotide sequence comprising the sequence of apolynucleotide of the invention in a computer readable medium; andscreening for at least one overlapping region between said firstpolynucleotide sequence and a second polynucleotide sequence.

[0205] In another preferred embodiment of the invention there isprovided a computer readable medium having stored thereon a memberselected from the group consisting of: a polynucleotide comprising thesequence of SEQ ID NO. 1 or 3; a polypeptide comprising the sequence ofSEQ ID NO. 2 or 4; a set of polynucleotide sequences wherein at leastone of said sequences comprises the sequence of SEQ ID NO. 1 or 3; a setof polypeptide sequences wherein at least one of said sequencescomprises the sequence of SEQ ID NO. 2 or 4; a data set representing apolynucleotide sequence comprising the sequence of SEQ ID NO. 1 or 3; adata set representing a polynucleotide sequence encoding a polypeptidesequence comprising the sequence of SEQ ID NO. 2 or 4; a polynucleotidecomprising the sequence of SEQ ID NO. 1 or 3; a polypeptide comprisingthe sequence of SEQ ID NO. 2 or 4; a set of polynucleotide sequenceswherein at least one of said sequences comprises the sequence of SEQ IDNO. 1 or 3; a set of polypeptide sequences wherein at least one of saidsequences comprises the sequence of SEQ ID NO. 2 or 4; a data setrepresenting a polynucleotide sequence comprising the sequence of SEQ IDNO. 1 or 3; a data set representing a polynucleotide sequence encoding apolypeptide sequence comprising the sequence of SEQ ID NO. 2 or 4. Afurther preferred embodiment of the invention provides a computer basedmethod for performing homology identification, said method comprisingthe steps of providing a polynucleotide sequence comprising the sequenceof SEQ ID NO. 1 or 3 in a computer readable medium; and comparing saidpolynucleotide sequence to at least one polynucleotide or polypeptidesequence to identify homology.

[0206] A still further preferred embodiment of the invention provides acomputer based method for performing homology identification, saidmethod comprising the steps of: providing a polypeptide sequencecomprising the sequence of SEQ ID NO. 2 or 4 in a computer readablemedium; and comparing said polypeptide sequence to at least onepolynucleotide or polypeptide sequence to identify homology.

[0207] A further embodiment of the invention provides a computer basedmethod for polynucleotide assembly, said method comprising the steps of:providing a first polynucleotide sequence comprising the sequence of SEQID NO. 1 or 3 in a computer readable medium; and screening for at leastone overlapping region between said first polynucleotide sequence and asecond polynucleotide sequence.

[0208] A further embodiment of the invention provides a computer basedmethod for performing homology identification, said method comprisingthe steps of: providing a polynucleotide sequence comprising thesequence of SEQ ID NO. 1 or 3 in a computer readable medium; andcomparing said polynucleotide sequence to at least one polynucleotide orpolypeptide sequence to identify homology.

[0209] A further embodiment of the invention provides a computer basedmethod for performing homology identification, said method comprisingthe steps of: providing a polypeptide sequence comprising the sequenceof SEQ ID NO. 2 or 4 in a computer readable medium; and comparing saidpolypeptide sequence to at least one polynucleotide or polypeptidesequence to identify homology.

[0210] A further embodiment of the invention provides a computer basedmethod for polynucleotide assembly, said method comprising the steps of:providing a first polynucleotide sequence comprising the sequence of SEQID NO. 1 or 3 in a computer readable medium; and screening for at leastone overlapping region between said first polynucleotide sequence and asecond polynucleotide sequence.

[0211] All publications and references, including but not limited topatents and patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

GLOSSARY

[0212] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein.

[0213] “Antibody(ies)” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

[0214] “Antigenically equivalent derivative(s)” as used hereinencompasses a polypeptide, polynucleotide, or the equivalent of eitherwhich will be specifically recognized by certain antibodies which, whenraised to the protein, polypeptide or polynucleotide according to theinvention, interferes with the immediate physical interaction betweenpathogen and mammalian host.

[0215] “Bispecific antibody(ies)” means an antibody comprising at leasttwo antigen binding domains, each domain directed against a differentepitope.

[0216] “Bodily material(s) means any material derived from an individualor from an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials.

[0217] “Disease(s)” means any disease caused by or related to infectionby a bacteria, including, for example, otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid.

[0218] “Fusion protein(s)” refers to a protein encoded by two, oftenunrelated, fused genes or fragments thereof. In one example, EP-A-0464discloses fusion proteins comprising various portions of constant regionof immunoglobulin molecules together with another human protein or partthereof. In many cases, employing an immunoglobulin Fc region as a partof a fusion protein is advantageous for use in therapy and diagnosisresulting in, for example, improved pharmacokinetic properties [see,e.g., EP-A 0232262]. On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

[0219] “Host cell(s)” is a cell which has been transformed ortransfected, or is capable of transformation or transfection by anexogenous polynucleotide sequence.

[0220] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asthe case may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SLAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

[0221] Parameters for polypeptide sequence comparison include thefollowing:

[0222] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0223] Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992)

[0224] Gap Penalty: 12

[0225] Gap Length Penalty: 4

[0226] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0227] Parameters for polynucleotide comparison include the following:

[0228] 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453(1970)

[0229] Comparison matrix: matches=+10, mismatch=0

[0230] Gap Penalty: 50

[0231] Gap Length Penalty: 3

[0232] Available as: The “gap” program from Genetics Computer Group,Madison Wis. These are the default parameters for nucleic acidcomparisons.

[0233] A preferred meaning for “identity” for polynucleotides andpolypeptides, as the case may be, are provided in (1) and (2) below.

[0234] (1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO: 1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO: 1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO: 1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO: 1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

[0235] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO: 1, y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and·is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO: 2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

[0236] By way of example, a polynucleotide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO: 1,that is it may be 100% identical, or it may include up to a certaininteger number of nucleic acid alterations as compared to the referencesequence such that the percent identity is less than 100% identity. Suchalterations are selected from the group consisting of at least onenucleic acid deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference polynucleotide sequenceor anywhere between those terminal positions, interspersed eitherindividually among the nucleic acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleic acid alterations for a given percent identity is determined bymultiplying the total number of nucleic acids in SEQ ID NO: 1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleic acids in SEQID NO: 1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

[0237] wherein n_(n) is the number of nucleic acid alterations, x_(n) isthe total number of nucleic acids in SEQ ID NO: 1, y is, for instance0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.,·is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

[0238] (2) Polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 50, 60, 70, 80,85, 90, 95, 97 or 100% identity to a polypeptide reference sequence ofSEQ ID NO: 2, wherein said polypeptide sequence may be identical to thereference sequence of SEQ ID NO: 2 or may include up to a certaininteger number of amino acid alterations as compared to the referencesequence, wherein said alterations are selected from the groupconsisting of at least one amino acid deletion, substitution, includingconservative and non-conservative substitution, or insertion, andwherein said alterations may occur at the amino- or carboxy-terminalpositions of the reference polypeptide sequence or anywhere betweenthose terminal positions, interspersed either individually among theamino acids in the reference sequence or in one or more contiguousgroups within the reference sequence, and wherein said number of aminoacid alterations is determined by multiplying the total number of aminoacids in SEQ ID NO: 2 by the integer defining the percent identitydivided by 100 and then subtracting that product from said total numberof amino acids in SEQ ID NO: 2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

[0239] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO: 2, y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and·is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

[0240] By way of example, a polypeptide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO: 2,that is it may be 100% identical, or it may include up to a certaininteger number of amino acid alterations as compared to the referencesequence such that the percent identity is less than 100% identity. Suchalterations are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein said alterations may occur atthe amino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence. Thenumber of amino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO: 2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO: 2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

[0241] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO: 2, y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., and·is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

[0242] “Immunologically equivalent derivative(s)” as used hereinencompasses a polypeptide, polynucleotide, or the equivalent of eitherwhich when used in a suitable formulation to raise antibodies in avertebrate, the antibodies act to interfere with the immediate physicalinteraction between pathogen and mammalian host.

[0243] “Immunospecific” means that characteristic of an antibody wherebyit possesses substantially greater affinity for the polypeptides of theinvention or the polynucleotides of the invention than its affinity forother related polypeptides or polynucleotides respectively, particularlythose polypeptides and polynucleotides in the prior art.

[0244] “Individual(s)” means a multicellular eukaryote, including, butnot limited to a metazoan, a mammal, an ovid, a bovid, a simian, aprimate, and a human.

[0245] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

[0246] “Organism(s)” means a (i) prokaryote, including but not limitedto, a member of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridium tetani, Clostridium botulinum, Treponema pallidum,Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon,including but not limited to Archaebacter, and (iii) a unicellular orfilamentous eukaryote, including but not limited to, a protozoan, afungus, a member of the genus Saccharomyces, Kluveromyces, or Candida,and a member of the species Saccharomyces ceriviseae, Kluveromyceslactis, or Candida albicans.

[0247] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

[0248] “Polypeptide(s)” refers to any peptide or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds. “Polypeptide(s)” refers to both short chains, commonlyreferred to as peptides, oligopeptides and oligomers and to longerchains generally referred to as proteins. Polypeptides may contain aminoacids other than the 20 gene encoded amino acids. “Polypeptide(s)”include those modified either by natural processes, such as processingand other post-translational modifications, but also by chemicalmodification techniques. Such modifications are well described in basictexts and in more detailed monographs, as well as in a voluminousresearch literature, and they are well known to those of skill in theart. It will be appreciated that the same type of modification may bepresent in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications. Modifications can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains, and theamino or carboxyl termini. Modifications include, for example,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

[0249] “Recombinant expression system(s)” refers to expression systemsor portions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

[0250] “Subtraction set” is one or more, but preferably less than 100,polynucleotides comprising at least one polynucleotide of the invention

[0251] “Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

[0252] The examples below are carried out using standard techniques,which are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples are illustrative, butdo not limit the invention.

Example 1

[0253] Strain Selection, Library Production and Sequencing

[0254] The polynucleotide having a DNA sequence given in Table 1 [SEQ IDNO: 1 or 3] was obtained from a library of clones of chromosomal DNA ofStreptococcus pneumoniae in E. coli. The sequencing data from two ormore clones containing overlapping Streptococcus pneumoniae DNAs wasused to construct the contiguous DNA sequence in SEQ ID NO: 1. Librariesmay be prepared by routine methods, for example: Methods 1 and 2 below.

[0255] Total cellular DNA is isolated from Streptococcus pneumoniae0100993 according to standard procedures and size-fractionated by eitherof two methods.

[0256] Method 1

[0257] Total cellular DNA is mechanically sheared by passage through aneedle in order to size-fractionate according to standard procedures.DNA fragments of up to 11 kbp in size are rendered blunt by treatmentwith exonuclease and DNA polymerase, and EcoRI linkers added. Fragmentsare ligated into the vector Lambda ZapII that has been cut with EcoRI,the library packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

[0258] Method 2

[0259] Total cellular DNA is partially hydrolyzed with a one or acombination of restriction enzymes appropriate to generate a series offragments for cloning into library vectors (e.g., RsaI, PalI, AluI,Bshl235I), and such fragments are size-fractionated according tostandard procedures. EcoRI linkers are ligated to the DNA and thefragments then ligated into the vector Lambda ZapII that have been cutwith EcoRI, the library packaged by standard procedures, and E.coliinfected with the packaged library. The library is amplified by standardprocedures.

Example 2

[0260] The Determination of Expression During Infection of a Gene fromStreptococcus pneumoniae

[0261] Excised lungs from a 48 hour respiratory tract infection ofStreptococcus pneumoniae 0100993 in the mouse is efficiently disruptedand processed in the presence of chaotropic agents and RNAase inhibitorto provide a mixture of animal and bacterial RNA. The optimal conditionsfor disruption and processing to give stable preparations and highyields of bacterial RNA are followed by the use of hybridisation to aradiolabelled oligonucleotide specific to Streptococcus pneumoniae 16SRNA on Northern blots. The RNAase free, DNAase free, DNA and proteinfree preparations of RNA obtained are suitable for Reverse TranscriptionPCR (RT-PCR) using unique primer pairs designed from the sequence ofeach gene of Streptococcus pneumoniae 0100993.

[0262] a) Isolation of Tissue Infected with Streptococcus pneumoniae0100993 from a Mouse Animal Model of Infection (Lungs)

[0263]Streptococcus pneumoniae 0100993 is grown either on TSA/5% horseblood plates or in AGCH medium overnight, 37° C., 5% CO2. Bacteria arethen collected and resuspended in phosphate-buffered saline to an A600of approximately 0.4. Mice are anaesthetized with isofluorane and 50 mlof bacterial suspension (approximately 2×105 bacteria) is administeredintranasally using a pipetman. Mice are allowed to recover and have foodand water ad libitum. After 48 hours, the mice are euthanized by carbondioxide overdose, and lungs are aseptically removed and snap-frozen inliquid nitrogen.

[0264] b) Isolation of Streptococcus pneumoniae 0100993 RNA fromInfected Tissue Samples

[0265] Infected tissue samples, in 2-ml cryo-strorage tubes, are removedfrom −80° C. storage into a dry ice ethanol bath. In a microbiologicalsafety cabinet the samples are disrupted up to eight at a time while theremaining samples are kept frozen in the dry ice ethanol bath. Todisrupt the bacteria within the tissue sample, 50-100 mg of the tissueis transfered to a FastRNA tube containing a silica/ceramic matrix(BIO101). Immediately, 1 ml of extraction reagents (FastRNA reagents,BIO101) are added to give a sample to reagent volume ratio ofapproximately 1 to 20. The tubes are shaken in a reciprocating shaker(FastPrep FP120, BIO101) at 6000 rpm for 20-120 sec. The crude RNApreparation is extracted with chloroform/isoamyl alcohol, andprecipitated with DEPC-treated/Isopropanol Precipitation Solution(BIO101). RNA preparations are stored in this isopropanol solution at−80° C. if necessary. The RNA is pelleted (12,000 g for 10 min.), washedwith 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10 min,and resuspended in 0.1 ml of DEPC-treated water, followed by 5-10minutes at 55° C. Finally, after at least 1 minute on ice, 200 units ofRnasin (Promega) is added.

[0266] RNA preparations are stored at −80° C. for up to one month. Forlonger term storage the RNA precipitate can be stored at the wash stageof the protocol in 75% ethanol for at least one year at −20° C.

[0267] Quality of the RNA isolated is assessed by running samples on 1%agarose gels. 1×TBE gels stained with ethidium bromide are used tovisualise total RNA yields. To demonstrate the isolation of bacterialRNA from the infected tissue 1×MOPS, 2.2M formaldehyde gels are run andvacuum blotted to Hybond-N (Amersham). The blot is then hybridised witha 32P-labelled oligonucletide probe, of sequence 5′AACTGAGACTGGCTTTAAGAGATTA 3′, [SEQ ID NO: 9] specific to 16S rRNA ofStreptococcus pneumoniae. The size of the hybridising band is comparedto that of control RNA isolated from in vitro grown Streptococcuspneumoniae 0100993 in the Northern blot. Correct sized bacterial 16SrRNA bands can be detected in total RNA samples which show degradationof the mammalian RNA when visualised on TBE gels.

[0268] c) The Removal of DNA from Streptococcus pneumoniae—Derived RNA

[0269] DNA was removed from 50 microgram samples of RNA by a 30 minutetreatment at 37° C. with 20 units of RNAase-free DNAaseI (GenHunter) inthe buffer supplied in a final volume of 57 microliters.

[0270] The DNAase was inactivated and removed by treatment with TRizolLS Reagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol. DNAase treated RNA was resuspended in 100 microliters of DEPCtreated water with the addition of Rnasin as described before.

[0271] d) The Preparation of cDNA from RNA Samples Derived from InfectedTissue

[0272] 3 microgram samples of DNAase treated RNA are reverse transcribedusing a SuperScript Preamplification System for First Strand cDNASynthesis kit (Gibco BRL, Life Technologies) according to themanufacturers instructions. 150 nanogram of random hexamers is used toprime each reaction. Controls without the addition of SuperScriptIIreverse transcriptase are also run. Both +/−RT samples are treated withRNaseH before proceeding to the PCR reaction

[0273] e) The Use of PCR to Determine the Presence of a Bacterial cDNASpecies

[0274] PCR reactions are set up on ice in 0.2 ml tubes by adding thefollowing components: 43 microliters PCR Master Mix (AdvancedBiotechnologies Ltd.); 1 microliter PCR primers (optimally 18-25basepairs in length and designed to possess similar annealingtemperatures), each primer at 10 mM initial concentration; and 5microliters cDNA.

[0275] PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600as follows: 2 minutes at 94° C., then 50 cycles of 30 seconds each at94° C., 50° C. and 72° C. followed by 7 minutes at 72° C. and then ahold temperature of 20° C. (the number of cycles is optimally 30-50 todetermine the appearance or lack of a PCR product and optimally 8-30cycles if an estimation of the starting quantity of cDNA from the RTreaction is to be made); 10 microliter aliquots are then run out on 1%1×TBE gels stained with ethidium bromide, with PCR product, if present,sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, LifeTechnologies). Alternatively if the PCR products are convenientlylabelled by the use of a labelled PCR primer (e.g. labelled at the 5′end with a dye) a suitable aliquot of the PCR product is run out on apolyacrylamide sequencing gel and its presence and quantity detectedusing a suitable gel scanning system (e.g. ABI Prism™ 377 Sequencerusing GeneScan™ software as supplied by Perkin Elmer).

[0276] RT/PCR controls may include +/− reverse transcriptase reactions,16S rRNA primers or DNA specific primer pairs designed to produce PCRproducts from non-transcribed Streptococcus pneumoniae 0100993 genomicsequences.

[0277] To test the efficiency of the primer pairs they are used in DNAPCR with Streptococcus pneumoniae 0100993 total DNA. PCR reactions areset up and run as described above using approx. 1 microgram of DNA inplace of the cDNA.

[0278] Primer pairs which fail to give the predicted sized product ineither DNA PCR or RT/PCR are PCR failures and as such are uninformative.Of those which give the correct size product with DNA PCR two classesare distinguished in RT/PCR: 1.Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR; and 2.Genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stronger signal in the +RT samples than the signal (if at allpresent) in −RT controls Based on these analyses it was discovered thatthis Streptococcus pneumoniae histidine kinase gene was transcribed invivo.

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidhaving at least: (a) 70% identity; (b) 80% identity; (c) 90% identity;or (d) 95% identity to the amino acid sequence of SEQ ID NO: 2 or 4 overthe entire length of SEQ ID NO: 2 or 4; (ii) an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 2 or 4, (iii) anisolated polypeptide which is the amino acid sequence of SEQ ID NO: 2 or4, and (iv) a polypeptide which is encoded by a recombinantpolynucleotide comprising the polyncleotide sequence of SEQ ID NO: 2 or3.
 2. An isolated polynucleotide selected from the group consisting of:(i) an isolated polynucleotide comprising a polynucleotide sequenceencoding a polypeptide that has at least (a) 70% identity; (b) 80%identity; (c) 90% identity; or (d) 95% identity; to the amino acidsequence of SEQ ID NO: 2 or 4, over the entire length of SEQ ID NO: 2 or4; (ii) an isolated polynucleotide comprising a polynucleotide sequencethat has at least: (a) 70% identity (b) 80% identity; (c) 90% identity;or (d) 95% identity; over its entire length to a polynucleotide sequenceencoding the polypeptide of SEQ ID NO: 2 or 4; (iii) an isolatedpolynucleotide comprising a nucleotide sequence which has at least: (a)70% identity; (b) 80% identity; (c) 90% identity; or (d) 95% identity;to that of SEQ ID NO: 1 or 3 over the entire length of SEQ ID NO: 1 or3; (iv) an isolated polynucleotide comprising a nucleotide sequenceencoding the polypeptide of SEQ ID NO: 2 or 4; (vi) an isolatedpolynucleotide which is the polynucleotide of SEQ ID NO: 1 or 3; (vi) anisolated polynucleotide obtainable by screening an appropriate libraryunder stringent hybridization conditions with a probe having thesequence of SEQ ID NO: 1 or 3 or a fragment thereof; (vii) an isolatedpolynucleotide encoding a mature polypeptide expressed by the Histidinekinase gene contained in Streptococcus pneumoniae; and (viii) apolynucleotide sequence complementary to said isolated polynucleotide of(i), (ii), (iii), (iv), (v), (vi) or (vii).
 3. An antibody antigenic toor immunospecific for the polypeptide of claim 1 .
 4. A method for thetreatment of an individual: (i) in need of enhanced activity orexpression of the polypeptide of claim 1 comprising the step of: (a)administering to the individual a therapeutically effective amount of anagonist to said polypeptide; or (b) providing to the individual anisolated polynucleotide comprising a polynucleotide sequence encodingsaid polypeptide in a form so as to effect production of saidpolypeptide activity in vivo; or (ii) having need to inhibit activity orexpression of the polypeptide of claim 1 comprising: (a) administeringto the individual a therapeutically effective amount of an antagonist tosaid polypeptide; or (b) administering to the individual a nucleic acidmolecule that inhibits the expression of a polynucleotide sequenceencoding said polypeptide; or (c) administering to the individual atherapeutically effective amount of a polypeptide that competes withsaid polypeptide for its ligand, substrate, or receptor.
 5. A processfor diagnosing or prognosing a disease or a susceptibility to a diseasein an individual related to expression or activity of the polypeptide ofclaim 1 in an individual comprising the step of: (a) determining thepresence or absence of a mutation in the nucleotide sequence encodingsaid polypeptide in the genome of said individual; or (b) analyzing forthe presence or amount of said polypeptide expression in a samplederived from said individual.
 6. A method for screening to identifycompounds that activate or that inhibit the function of the polypeptideof claim 1 which comprises a method selected from the group consistingof: (a) measuring the binding of a candidate compound to the polypeptideor to the cells or membranes bearing the polypeptide or a fusion proteinthereof by means of a label directly or indirectly associated with thecandidate compound; (b) measuring the binding of a candidate compound tothe polypeptide or to the cells or membranes bearing the polypeptide ora fusion protein thereof in the presence of a labeled competitor; (c)testing whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide, using detection systemsappropriate to the cells or cell membranes bearing the polypeptide; (d)mixing a candidate compound with a solution containing a polypeptide ofclaim 1 , to form a mixture, measuring activity of the polypeptide inthe mixture, and comparing the activity of the mixture to a standard;(e) detecting the effect of a candidate compound on the production ofmRNA encoding said polypeptide and said polypeptide in cells, using forinstance, an ELISA assay, or (f) (1) contacting a composition comprisingthe polypeptide with the compound to be screened under conditions topermit interaction between the compound and the polypeptide to assessthe interaction of a compound, such interaction being associated with asecond component capable of providing a detectable signal in response tothe interaction of the polypeptide with the compound; and (2)determining whether the compound interacts with and activates orinhibits an activity of the polypeptide by detecting the presence orabsence of a signal generated from the interaction of the compound withthe polypeptide.
 7. An agonist or an antagonist of the activity orexpression polypeptide of claim 1 .
 8. An expression system comprising apolynucleotide capable of producing a polypeptide of claim 1 when saidexpression system is present in a compatible host cell.
 9. A host cellcomprising the expression system of claim 8 or a membrane thereofexpressing a polypeptide selected from the group consisting of: (i) anisolated polypeptide comprising an amino acid sequence selected from thegroup having at least: (a) 70% identity; (b) 80% identity; (c) 90%identity; or (d) 95% identity to the amino acid sequence of SEQ ID NO: 2or 4 over the entire length of SEQ ID NO: 2 or 4; (ii) an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4;(iii) an isolated polypeptide which is the amino acid sequence of SEQ IDNO: 2 or 4, and (iv) a polypeptide which is encoded by a recombinantpolynucleotide comprising the polynucleotide sequence of SEQ ID NO: 2 or3.
 10. A process for producing a polypeptide selected from the groupconsisting of: (i) an isolated polypeptide comprising an amino acidsequence selected from the group having at least: (a) 70% identity; (b)80% identity; (c) 90% identity; or (d) 95% identity to the amino acidsequence of SEQ ID NO: 2 or 4 over the entire length of SEQ ID NO: 2 or4; (ii) an isolated polypeptide comprising the amino acid sequence ofSEQ ID NO: 2 or 4; (iii) an isolated polypeptide which is the amino acidsequence of SEQ ID NO: 2 or 4, and (iv) a polypeptide which is encodedby a recombinant polynucleotide comprising the polynucleotide sequenceof SEQ ID NO: 2 or 3, comprising the step of culturing a host cell ofclaim 9 under conditions sufficient for the production of saidpolypeptide.
 11. A process for producing a host cell comprising theexpression system of claim 8 or a membrane thereof expressing apolypeptide selected from the group consisting of: (i) an isolatedpolypeptide comprising an amino acid sequence selected from the grouphaving at least: (a) 70% identity; (b) 80% identity; (c) 90% identity;or (d) 95% identity to the amino acid sequence of SEQ ID NO: 2 or 4 overthe entire length of SEQ ID NO: 2 or 4; (ii) an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO: 2 or 4; (iii) anisolated polypeptide which is the amino acid sequence of SEQ ID NO: 2 or4, and (iv) a polypeptide which is encoded by a recombinantpolynucleotide comprising the polynucleotide sequence of SEQ ID NO: 2 or3, said process comprising the step of transforming or transfecting acell with an expression system comprising a polynucleotide capable ofproducing said polypeptide of (i), (ii), (iii) or (iv) when saidexpression system is present in a compatible host cell such the hostcell, under appropriate culture conditions, produces said polypeptide of(i), (ii), (iii) or (iv).
 12. A host cell produced by the process ofclaim 11 or a membrane thereof expressing a polypeptide selected fromthe group consisting of: (i) an isolated polypeptide comprising an aminoacid sequence selected from the group having at least: (a) 70% identity;(b) 80% identity; (c) 90% identity; or (d) 95% identity to the aminoacid sequence of SEQ ID NO: 2 or 4 over the entire length of SEQ ID NO:2 or 4; (ii) an isolated polypeptide comprising the amino acid sequenceof SEQ ID NO: 2 or 4; (iii) an isolated polypeptide which is the aminoacid sequence of SEQ ID NO: 2 or 4, and (iv) a polypeptide which isencoded by a recombinant polynucleotide comprising the polynucleotidesequence of SEQ ID NO: 2 or
 3. 13. A computer readable medium havingstored thereon a member selected from the group consisting of: apolynucleotide comprising the sequence of SEQ ID NO. 1 or 3; apolypeptide comprising the sequence of SEQ ID NO. 2 or 4; a set ofpolynucleotide sequences wherein at least one of said sequencescomprises the sequence of SEQ ID NO. 1 or 3; a set of polypeptidesequences wherein at least one of said sequences comprises the sequenceof SEQ ID NO. 2 or 4; a data set representing a polynucleotide sequencecomprising the sequence of SEQ ID NO. 1 or 3; a data set representing apolynucleotide sequence encoding a polypeptide sequence comprising thesequence of SEQ ID NO. 2 or 4; a polynucleotide comprising the sequenceof SEQ ID NO. 1 or 3; a polypeptide comprising the sequence of SEQ IDNO. 2 or 4; a set of polynucleotide sequences wherein at least one ofsaid sequences comprises the sequence of SEQ ID NO. 1 or 3; a set ofpolypeptide sequences wherein at least one of said sequences comprisesthe sequence of SEQ ID NO. 2 or 4; a data set representing apolynucleotide sequence comprising the sequence of SEQ ID NO. 1 or 3; adata set representing a polynucleotide sequence encoding a polypeptidesequence comprising the sequence of SEQ ID NO. 2 or
 4. 14. A computerbased method for performing homology identification, said methodcomprising the steps of providing a polynucleotide sequence comprisingthe sequence of SEQ ID NO. 1 or 3 in a computer readable medium; andcomparing said polynucleotide sequence to at least one polynucleotide orpolypeptide sequence to identify homology.
 15. A further embodiment ofthe invention provides a computer based method for polynucleotideassembly, said method comprising the steps of: providing a firstpolynucleotide sequence comprising the sequence of SEQ ID NO. 1 or 3 ina computer readable medium; and screening for at least one overlappingregion between said first polynucleotide sequence and a secondpolynucleotide sequence.
 16. An isolated polynucleotide selected formthe group consisting of: (a) an isolated polynucleotide comprising anucleotide sequence which has at least 70%, 80%, 90%, 95%, 97% identityto SEQ ID NO: 3 over the entire length of SEQ ID NO: 3; (b) an isolatedpolynucleotide comprising the polynucleotide of SEQ ID NO: 3; (c) thepolynucleotide of SEQ ID NO: 3; or (d) an isolated polynucleotidecomprising a nucleotide sequence encoding a polypeptide which has atleast 70%, 80%, 90%, 95%, 97-99% identity to the amino acid sequence ofSEQ ID NO: 4, over the entire length of SEQ ID NO:
 4. 17. A polypeptideselected from the group consisting of: (a) a polypeptide which comprisesan amino acid sequence which has at least 70%, 80%, 90%, 95%, 97-99%identity to that of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4;(b) a polypeptide which has an amino acid sequence which is at least70%, 80%, 90%, 95%, 97-99% identity to the amino acid sequence of SEQ IDNO: 4 over the entire length of SEQ ID NO: 4; (c) a polypeptide whichcomprises the amino acid of SEQ ID NO: 4; (d) a polypeptide which is thepolypeptide of SEQ ID NO: 4; (e) a polypeptide which is encoded by apolynucleotide comprising the sequence contained in SEQ ID NO: 3.